Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Abstract

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8(+) T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.

Figure 1

Transcription and expression of Stx11 in cytotoxic lymphocytes. NK cells or CD8⁺ T cells were isolated from PBMCs by negative selection. (A) cDNA was prepared from cells and cell lines. RT-PCR was performed with gene-specific primers as indicated. (B) Stx11 expression relative to actin control was measured by immunoblotting in lysates derived from purified cell CD8⁺ T-cell or NK-cell populations, as well as the NK-cell line NK92. Arrowheads indicate bands corresponding to actin and Stx11 immunoreactivity.

Figure 2

Methodological platform and relative degranulating capacity of lymphocyte subsets. (A-D) Freshly isolated, resting PBLs alone or mixed with target cells and mAbs as indicated were incubated for 2 hours at 37°C. (A,B) Thereafter, cells were stained with fluorochrome-conjugated anti-CD3, -CD56, and -CD107a mAbs and analyzed by flow cytometry. (A) Lymphocytes were gated on forward scatter/side scatter plots, followed by gating of CD3⁺, CD3⁻CD56⁺, and CD3⁻CD56⁻ cell subsets. (B) Induced CD107a surface expression (ΔCD107a⁺) was calculated as the percentage of CD107a⁺ cells after indicated stimulation subtracted from the percentage of CD107a⁺ cells after incubation of PBLs alone. For gating on CD3⁺ cells, a fluorochrome-conjugated anti-CD3 mAb was used. Values represent the mean (± SD) of 6 donors. (C,D) After indicated stimulation, cells were stained with fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD62L, anti-CD107a, and anti-CCR7 mAbs. (C) T cells were gated on forward scatter/side scatter plots for lymphocytes (R1 gate), followed by forward scatter/forward area plots (R2 gate), and gated on CD3⁺CD14⁻ cells (R3 gate). Contour plots show CD3⁺CD14⁻ T-cell populations (R3 gate) overlayed with dots representing CD3⁺CD14⁻CD107a⁺ T cells (R4 gate). The frequencies of degranulating T cells are indicated. Numbers indicate the percentage of degranulating T cells within each quadrant. (D) Induced CD107a surface expression was determined for indicated CD3⁺CD14⁻ T-cell subsets. Values represent the mean (± SD) of 5 donors. Numbers indicate mean frequencies of CD3⁺CD14⁻ T-cell subsets and their SD. (E) Resting PBLs were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16-, anti-CD56, and anti-CD62L mAbs, followed by fixation, permeabilization, and intracellular staining with anti-CD107a, antiperforin, antigranzyme A, or anti-CD63 mAbs. Values represent relative mean fluorescence intensity (R-MFI), where MFI values for indicated staining have been subtracted for MFI values for isotype control mAbs. Values represent mean (± SD) of 5 donors.

Figure 3

Expression of perforin and Stx11 in FHL patients. (A) PBLs were stained with fluorochrome-conjugated anti-CD3, and anti-CD56 mAbs. Thereafter, cells were fixed, permeabilized, stained with antiperforin mAb, and analyzed by flow cytometry. Histograms depict perforin expression in CD3⁻CD56⁺ NK cells. (B) Quantification of perforin staining by flow cytometry. Values represent relative mean fluorescence of perforin in CD3⁻CD56⁺ NK cells as percent of adult control cells. The error bar indicates the SD of normalized perforin expression for 12 healthy controls. (C) Stx11 expression relative to actin control was measured by immunoblotting. Arrowheads indicate bands corresponding to actin and Stx11 immunoreactivity.

Figure 4

Abrogation of degranulation and cytotoxicity by resting PBLs from FHL4 patients. (A) Resting PBLs from healthy adult and infant donors plus FHL2, FHL3, and FHL4 patients were evaluated for cytotoxicity toward K562 cells in 4-hour ⁵¹Cr release assays. Lytic units were calculated from specific lysis values. (B) Resting PBLs from adult and infant donors were stained with anti-CD3 and -CD56 mAbs and analyzed by flow cytometry. The percentage of indicated lymphocyte subsets was determined. Bars indicate the mean (±SD) of 7 donors. (C-E) Resting PBLs were incubated alone or with target cells as indicated for 2 hours at 37°C. Thereafter, cells were stained with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. (C) Lymphocytes were gated on forward scatter/side scatter plots, followed by gating on CD3 versus CD56 plots. Profiles show CD56 versus CD107a mAb staining of CD3⁻CD56⁺ NK cells. (D) Induced CD107a surface expression on CD3⁻CD56⁺ NK cells after indicated stimulation was plotted. Lines represent mean values. (E) Induced CD107a surface expression on lymphocytes after indicated stimulation was plotted. Lines represent mean values. (F) Resting PBLs from healthy adults or infant donors were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD62L, and anti-CCR7 mAbs. Values represent the mean (±SD) percentage of indicated CD3⁺CD14⁻ T-cell subsets.

Figure 5

Perforin expression in adult and cord blood NK cells and T cells. Cells from adult peripheral and cord blood donors were stained with fluorochrome-conjugated anti-CD3 and -CD56 mAbs, followed by fixation, permeabilization, and intracellular staining with antiperforin mAb. Lymphocytes were gated on forward scatter/side scatter plots, followed by gating on CD3⁺ and CD3⁻ cells. Profiles show (A) CD56 versus perforin mAb staining of CD3⁺ T cells and (B) CD56 versus perforin mAb staining of CD3⁻ cells.

Figure 6

Polarization of secretory lysosomes in Stx11-deficient resting NK cells. Resting NK cells from a FHL4 patient mixed with K562 target cells. Cells were fixed, permeabilized, and stained with antiperforin mAb (red) and DAPI (blue). Left panel shows a differential interference contrast (DIC) image. The right panel shows overlay of nuclear staining with DAPI and antiperforin mAb. Scales represent 5 μm.

Figure 7

IL-2 partially restores degranulation and cytotoxicity by PBLs from FHL4 patients. (A-E) PBLs from healthy adult and infant donors, plus FHL2, FHL3, and FHL4 patients were stimulated with 400 IU/mL IL-2 for 72 hours. (A) IL-2–stimulated PBLs were evaluated for cytotoxicity toward K562 cells in 4-hour ⁵¹Cr release assays. Lytic units were calculated from specific lysis values. (B) PBLs were maintained in medium or medium with IL-2 added for the indicated times. Thereafter, PBLs were incubated alone or with K562 cells as indicated for 2 hours at 37°C. Cells were stained with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. Induced CD107a surface expression on CD3⁻CD56⁺ NK cells after incubation with K562 cells relative to PBLs alone was plotted as a function of stimulation time with IL-2. (C-E) PBLs stimulated with IL-2 for 72 hours were incubated alone or with target cells as indicated for 2 hours at 37°C. Thereafter, cells were stained with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. (C) Lymphocytes were gated on forward scatter/side scatter plots, followed by gating on CD3 versus CD56 plots. Profiles show CD56 versus CD107a mAb staining of CD3⁻CD56⁺ NK cells. (D) Induced CD107a surface expression on CD3⁻CD56⁺ NK cells after indicated stimulation was plotted. Lines represent mean values. (E) Induced CD107a surface expression on lymphocytes after indicated stimulation was plotted. Lines represent mean values.