The functional basis for hemophagocytic lymphohistiocytosis in a patient with co-inherited missense mutations in the perforin (PFN1) gene

Abstract

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated ( approximately 45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure-function relationships for lymphocyte perforin.

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. The data points are shown as the mean ± SD of triplicate samples and are representative of three similar assays. The Western blot (right) shows truncation of T224W perforin expressed in two independent transfection experiments (T224W-1 and T224W-2) compared with WT and T224R perforin.

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 were stained as in A, after degranulation was induced by transient incubation with TNP-labeled target cells. Magnification, 400×.

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 vector-expressing cells (GFP). (B) Perforin-dependent ⁵¹Cr release from TNP-labeled Jurkat cells coincubated for 4 h in the presence of anti-TNP IgE with RBL cells stably expressing WT or G428E perforin. The data points are shown as the mean ± SD of three independent experiments. The Western blot (right) shows that G428E comigrates with WT perforin. GFP is the empty vector control. (C) RBL cells stably overexpressing WT or G428E perforin or the empty vector (GFP) were lysed and fractionated on a Percoll density gradient. Fractions were then analyzed for their perforin content by Western blotting and their β-hexosaminidase activity (as described in Materials and Methods).

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 (+) of 1 mM CaCl₂. The total input of perforin in each case is shown as C.