Human immunodeficiency-causing mutation defines CD16 in spontaneous NK cell cytotoxicity

Abstract

The Fc receptor on NK cells, FcγRIIIA (CD16), has been extensively studied for its role in mediating antibody-dependent cellular cytotoxicity (ADCC). A homozygous missense mutation in CD16 (encoding a L66H substitution) is associated with severe herpesvirus infections in rare patients. Here, we identified a new patient with this CD16 mutation and compared the patient's NK cells to those of the originally reported patient. Patients with the L66H mutation had intact ADCC, but deficient spontaneous NK cell cytotoxicity and decreased surface expression of CD2, a coactivation receptor. Mechanistic studies in a human NK cell line, NK-92, demonstrated that CD16 expression correlated with CD2 surface levels and enabled killing of a melanoma cell line typically resistant to CD16-deficient NK-92 cells. An association between CD16 and CD2 was identified biochemically and at the immunological synapse, which elicited CD16 signaling after CD2 engagement. Stable expression of CD16 L66H in NK-92 cells recapitulated the patient phenotype, abrogating association of CD16 with CD2 as well as CD16 signaling after CD2 ligation. Thus, CD16 serves a role in NK cell-mediated spontaneous cytotoxicity through a specific association with CD2 and represents a potential mechanism underlying a human congenital immunodeficiency.

Figure 1. Point mutation in CD16 results in CD16 B73.1 epitope loss and decreased natural killing without affecting ADCC.

(A) FACS analysis of CD3^–CD56⁺ NK cells from a control donor and patient 1 for CD16 3G8 and CD16 B73.1 epitopes. (B) Sequence analysis of CD16 within the region corresponding to the B73.1 epitope in a normal donor and patient 1. (C) NK cell lytic units (LU₂₀) against K562 cells for patient 1 and control (n = 3 independent experiments). (D) ADCC assay of patient 1 or control PBMCs against Raji cells in the presence or absence of rituximab (representative of n = 3 independent experiments).

Figure 2. Model of mutant CD16 structure.

(A) Rendering of the published wild-type CD16 structure. Yellow, membrane-proximal Ig-like domain; blue, membrane-distal Ig-like domain; black, B73.1 epitope loop. (B) Predicted CD16 L66H mutant structure. Purple, proximal domain; green, membrane-distal domain; red, B73.1 epitope loop. (C) Overlay of wild-type and L66H CD16 structures. Orange arrow denotes L66H abnormality.

Figure 3. NK cell phenotype of L66H patients.

PBMCs were gated on CD3^–CD56⁺ NK cells from patient 1 (A and B) or patient 2 (C and D). (A) FACS analysis for the indicated NK cell markers (thick gray line, patient 1; black filled regions, control donor; thin black line, isotype control), representative of 3 independent experiments from 3 independent blood draws. (B) Quantitative analysis of MFI for each marker relative to the isotype control. *P < 0.05, paired Student’s t test. (C) FACS analysis for the indicated NK cell markers (thick gray line, patient 2; black filled regions, control donor; thin black line, isotype control). Only 1 sample was available, and although a relatively small population of CD16 NK cells overall was noted, they were 3G8⁺B73.1^–. (D) Quantitative analysis of MFI for each marker relative to the isotype control.

Figure 4. Phenotype of NK-92 and CD16.NK-92 NK cell lines.

(A) FACS analysis of the indicated NK cell markers (thick gray line, NK-92; black filled regions, CD16.NK-92; thin black line, isotype control), representative of 3 independent experiments. (B) Quantitative analysis of MFI for each marker relative to isotype over at least 3 independent experiments. *P < 0.05, paired Student’s t test.

Figure 5. Functional activity of NK-92 cell lines.

Cytotoxic activity was assessed using 4-hour ⁵¹Cr-release assays. (A) NK-92 or CD16.NK-92 against K562 target cells (n = 5 independent experiments). ADCC assays against Raji cells using added rituximab for (B) NK-92 and (C) CD16.NK-92 cells, in which only the latter demonstrated activity (n = 4 independent experiments). Spontaneous cytotoxicity against mel1106 cells in the presence (D) and absence (E) of fetal calf serum in NK-92 and CD16.NK-92 cells (n = 5 independent assays). (F) Addition of anti-CD2 blocking mAb, but not anti-CD56 or nonspecific mIgG, abrogated CD16.NK-92 cytotoxicity against mel1106 cells (n = 3 independent assays). *P < 0.05, paired Student’s t test (comparisons of individual points); **P < 0.05, Wilcoxon signed-rank test (comparisons across independent assays).

Figure 6. CD16 does not improve conjugate formation, but accumulates at the mel1106:NK cell synapse.

(A) Conjugation assay, expressed as percent NK cells conjugated to target cells over 4 hours (n = 3 independent experiments). No significance was identified using Wilcoxon signed-rank test (independent assays) or Student’s t test (individual points). (B) Representative fixed-cell images of actin (yellow) or CD16 (red) in unconjugated NK-92 cells, unconjugated CD16.NK-92 cells, and CD16.NK-92 cells conjugated with mel1106 cells. Scale bars: 5 μm. 5 conjugates or control cells were analyzed from each of 3 independent experiments (n = 15 per condition). (C) CD16 quantitation (mean ± SD) at the mel1106:NK cell synapse compared with unconjugated effectors, using regions of equal size (n = 15 per condition). *P < 0.05, paired Student’s t test. Subtraction of unconjugated CD16 values from CD16 accumulated at the immune synapse demonstrated a mean positive value.

Figure 7. The CD16.L66H.NK-92 cell line phenotypically and functionally recapitulates patient NK cells.

(A) FACS analysis of NK-92, CD16.NK-92, and CD16.L66H.NK-92 cell lines using CD16 mAbs, 3G8, and B73.1 (representative of n = 5). (B) Spontaneous cytotoxicity against K562 target cells (n = 3 independent experiments; no significant differences). (C) Spontaneous cytotoxicity against mel1106 cells. **P < 0.05, Wilcoxon signed-rank test (n = 3 independent experiments). (D) FACS analysis of surface CD2. Corrected MFI (relative to isotype control) was significantly greater in CD16.NK-92 cells. *P < 0.05, paired Student’s t test (n = 5 individual experiments). (E) Western blot analysis of CD2 expression in whole cell lysates. Actin was used as a loading control on the same membrane following stripping and reprobing (representative of 5 independent experiments).

Figure 8. CD16 associates with CD2 and engages CD16 signaling machinery.

(A) Confocal immunofluorescence for CD16 (red) localization relative to CD2 (green) and CD56 (purple) at the immune synapse in CD16.NK-92 and CD16.L66H.NK-92 conjugated with mel1106 cells. Images are representative of n = 30 across 3 independent experiments. Scale bars: 5 μm. (B) Quantitative analysis of percent CD16 colocalized with CD2 (circles) or CD56 (squares) at the immune synapse of CD16.NK-92:mel1106 (filled symbols) or CD16.L66H.NK-92:mel1106 (open symbols). Each point represents percent colocalization from a single conjugate; horizontal lines denote mean; error bars denote SD. ***P < 0.0001, unpaired Student’s t test; n = 30. (C) CD2 immunoprecipitation from the indicated cell lines and Western blot analysis for CD2, CD16, and TCRζ, all using the same membrane after stripping and reprobing (representative of n = 5). (D) Western blot analysis of phosphorylated TCRζ in TCRζ immunoprecipitates from CD16.NK-92 and CD16.L66H.NK-92 cells activated with either anti-CD2 or anti-CD56 Ab and then goat anti-mouse IgG. Total TCRζ is shown as a loading control. Representative of 3 experiments.