Human natural killer (NK) cells are defined as CD5

Berrien-Elliott, Melissa M

Dixon, Amera L

Ebrahim, Tasneem A.M.

Fehniger, Todd A

Gunesch, Justin T

Hegewisch-Solloa, Everardo

Kumar, Tejas

Mace, Emily M

Tatineni, Swetha

English

Digital Commons@Becker

Figures and figure supplementsCD56 regulates human NK cell cytotoxicity through Pyk2Justin T Gunesch et alGunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 1 of 16RESEARCH ARTICLEFigure 1. Validation of CD56 deletion in human NK cell lines and characterization of CD56 and its polysialation in human NK cells. (A) Western blotanalysis of CD56 from wild-type (WT) and CD56-knockout (KO) YTS (left) and NK92 (right) cell lines or primary human NK cells with actin as a loadingFigure 1 continued on next pageGunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 2 of 16Research article Cell Biology Immunology and InflammationFigure 1 continuedcontrol. (B) Flow cytometry analysis of CD56 expression in NK92 or YTS WT (filled histogram, dark grey) or CD56-KO (filled histogram, light grey) cellscompared to unstained cells (dashed line). (C) NK92 or YTS cells were treated with PNGase F to remove polysialic acid. Following treatment, lysateswere separated by SDS-PAGE and CD56 or actin as a loading control were detected by Western blotting. (D) NK cell lines (left) or Jurkat or Raji cells asa positive control (right) were treated with PI-PLC to cleave GPI anchored proteins from the cell surface. PI-PLC activity was confirmed by cleavage ofGPI-anchored CD55 (right). All data shown are representative of 3 technical replicates performed on different days.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 3 of 16Research article Cell Biology Immunology and InflammationFigure 1—figure supplement 1. Expression of PSA-NCAM on human NK cell lines. NK92 or YTS cell line lysateswere pre-treated with PNGase F to remove PSA-NCAM through cleavage of N-linked glycans. Following enzymetreatment, lysates from treated and untreated conditions were separated by SDS-PAGE and Western blotting withanti-PSA-NCAM antibody or actin as a loading control was performed.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 4 of 16Research article Cell Biology Immunology and InflammationFigure 2. CD56 deletion abrogates NK92 cytotoxic function and delays YTS cytotoxicity. 51Cr-release assays wereperformed using NK92 (A, C, D) or YTS (B, C) WT and CD56-KO cell lines as effectors against susceptible targets.(A) 4 hr assays were performed with NK92 cell lines using K562 (left) or 721.221 (right) target cells. (B) 4 hr assayswere performed with YTS cell lines using 721.221 (left) or KT86 (right) target cells. (C) 1 hr 51Cr-release assays wereperformed using NK92 (left) or YTS (right) cells as effectors. (D) CD56 (NCAM-140) was re-expressed in NK92CD56-KO cells and these cells, NK92 or NK92 CD56-KO cells were used for 4 hr cytotoxicity assays against K562(left) or 721.221 (right) target cells. (E) CD56-KO NK92 cells were transfected with chimeric CD56 constructs fusedto an mApple fluorescent reporter as described in Materials and methods. Flow cytometry was used to confirmthe expression of CD56 and/or mApple. (F) Cytotoxicity assays were performed with chimeric cell lines using K562cells as targets. Mean ± S.D. of three independent experiments pooled. **p<0.01, ***p<0.001, ****p<0.0001 byOrdinary one-way ANOVA with multiple corrections test or unpaired student t-test with Welch’s correction. DECD:chimeric construct lacking extracellular domain, DICD: chimeric construct lacking intracellular domain.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 5 of 16Research article Cell Biology Immunology and InflammationFigure 2—figure supplement 1. Phenotyping of NK cell lines by flow cytometry. NK92 and YTS WT and CD56-KO cells were analyzed for expressionof cell surface receptors and intracellular effector molecules using five panels as described in Materials and methods. Effector functions were evaluatedin the presence (activated) or absence (rest) of activation by PMA and ionomycin. Mean fluorescence intensity was measured and % positive cells basedon fluorescence minus one gating was also calculated. Shown is one representative of 3 independent experiments.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 6 of 16Research article Cell Biology Immunology and InflammationFigure 2—figure supplement 2. Re-expression of CD56 and PSA-NCAM in reconstituted NK92 cell lines. CD56 (left) or PSA-NCAM (right) weredetected by flow cytometry on WT (solid line), CD56-KO (dashed line) or CD56-KO cells reconstituted with NCAM140 (dot-dashed line). Isotypeantibody was used as a negative control (solid filled histogram). Shown is one representative of two independent repeats.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 7 of 16Research article Cell Biology Immunology and InflammationFigure 3. CD56 expression is required for exocytosis of NK92 cells. (A) WT NK92, CD56-KO or CD56-KO reconstituted cells were incubated for 60–90min on plates pre-coated with 10 mg/ml of anti-CD18 and anti-NKp30 antibodies. Supernatant was collected and granzyme A secretion was measuredFigure 3 continued on next pageGunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 8 of 16Research article Cell Biology Immunology and InflammationFigure 3 continuedby a BLT esterase assay. Secretory potential was measured as a readout of the % maximum of granzyme A activity in the supernatant. (B) WT NK92,CD56-KO or CD56-KO reconstituted cells were incubated for 1–2 hr on plates pre-coated with 10 mg/ml of anti-CD18 and anti-NKp30 antibodies. Cellswere harvested and degranulation was measured by CD107a expression using flow cytometry. (C) WT NK92, CD56-KO or CD56-KO reconstituted cellswere co-cultured with 721.221 target cells. Cells were harvested and CD107a expression was measured by flow cytometry. For co-culture experimentsthe average background of media only was subtracted from samples. Mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVAwith Tukey’s multiple comparisons post-hoc test. (D) WT YTS and CD56-KO cells were incubated with 721.221 target cells at a 2:1 ratio for 22 hr.Supernatant was collected and used in a human IFN gamma ELISA. ****p<0.0001 by unpaired student t-test with Welch’s correction. All data arerepresentative of 3 independent experiments performed in duplicate or triplicate.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 9 of 16Research article Cell Biology Immunology and InflammationFigure 4. Impaired immune synapse formation in NK92 CD56-KO cells. WT or CD56-KO effector cells were cultured at a 2:1 ratio with K562 target cellsfor 45 min then fixed, immunostained and visualized by confocal microscopy. (A) Representative images from >30 cells from three independentFigure 4 continued on next pageGunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 10 of 16Research article Cell Biology Immunology and InflammationFigure 4 continuedexperiments immunostained as indicated. (B) Integrated intensity of actin at the immune synapse for WT or CD56-KO cells. Data arerepresentative from one experiment performed three times. n = 30, 39. NS = not significant by unpaired t-test. (C) MTOC to synapse distance (mm)calculated from WT or CD56-KO conjugates. n = 70, 83. Data pooled from three independently replicated experiments. (D) Mean granule to centroiddistance for WT or CD56-KO conjugates. Each data point represents the mean distance granule to centroid distance from one conjugate. n = 33, 45from one representative experiment of >3 experiments. NS = not significant by unpaired T test with Welch’s correction. (E) WT or CD56-KO NK92effector cells were differentially labeled then conjugated at a 2:1 ratio with K562 target cells for the times indicated then fixed and analyzed by flowcytometry. The frequency of NK92-K562 conjugates was calculated for each timepoint. Each point represents a single experiment performed ondifferent days in triplicate (n = 4 replicates). Error bars indicate mean ± SD; *p<0.05 by Mann-Whitney test.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 11 of 16Research article Cell Biology Immunology and InflammationFigure 5. Phosphorylation of Pyk2 is decreased in NK92 CD56-KO cells upon activating receptor ligation. (A) NK92WT, CD56-KO or CD56 reconstituted (KO+CD56) cells were incubated for 25–30 min on plates pre-coated with 10mg/ml of anti-CD18 and anti-NKp30 antibodies. Cells were permeabilized and immunostained for pPyk2 Y402 thendata were acquired by flow cytometry. Relative fluorescent intensity (RFI) of pPyk2 was calculated based upon theintensity of the WT NK92 condition. Shown are the pooled data from three independent experiments. (B) WT orCD56-KO NK92 or YTS cells were permeabilized and immunostained for pPyk2 Y402 then data were acquired byflow cytometry. Shown is pooled data from 2 (YTS) or 3 (NK92) independent experiments. **p<0.01 by one-wayANOVA with multiple comparisons. (C) 4 hr 51Cr assays were performed with WT (black) or CD56-KO (red) NK92cells as effectors. Assays were performed in the presence of Pyk2 inhibitor PF431396 or vehicle control (DMSO)following brief pre-incubation of effectors with inhibitor. Shown are representative data from three independentrepeats. (D) Pooled data from the 10:1 effector to target cell ratio of the experiments described in (C) normalizedto the WT YTS condition without inhibitor. ***p<0.001 by one-way ANOVA with multiple comparisons. (E)Representative confocal microscopy images of WT or CD56-KO NK92 effectors conjugated to K562 target cells inthe presence of non-blocking CD56 antibody then fixed and immunostained for perforin, pPyk2 Y402 and actin. (F)Fluorescent intensity of pPYK2 Y402 at the immune synapse of WT or CD56-KO effector cells. n = 24, 28 from onerepresentative experiment of 3 independent repeats.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 12 of 16Research article Cell Biology Immunology and InflammationFigure 5—figure supplement 1. Detection of total Pyk2 in WT and CD56-KO cell lines. WT (top) or CD56-KO (bottom) NK92 cells were conjugated toK562 targets then fixed, permeabliized and immunostained for tubulin, Pyk2, actin (phalloidin) and CD56. Images were acquired on a spinning diskconfocal microscope. Shown is one representative of 60 cells from two independent technical replicates.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 13 of 16Research article Cell Biology Immunology and InflammationFigure 6. CD56-deficient primary NK cells retain lytic function. Primary NK cells were isolated and allowed to rest overnight in the presence of low-doseIL-15 prior to delivery of CD56 CRISPR-Cas9. Cells were further expanded in the presence of 25 ng/ml IL-15 for 15 days and cytotoxicity against K562Figure 6 continued on next pageGunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 14 of 16Research article Cell Biology Immunology and InflammationFigure 6 continuedtargets was measured. (A) Representative FACS plot of CD56-deficient (blue) or control primary cells (red) after 15 days of IL-15 expansion. Shown alsois the fluorescence minus one control (yellow). (B) K562 target cell lysis by primary NK cells shown in (A). (C) Control or CD56-deficient NK cells fromthree healthy donors were incubated for 1 week after CD56 CRISPR-Cas9 delivery in 25 ng/mL IL-15 then cells were isolated by FACS and cultured foran additional 8 days and the MFI of CD56 was measured by flow cytometry. (D) Specific lysis of K562 target cells by isolated and expanded CD56brightNK cells from the three healthy donors shown in (C). (E) Primary NK cells were incubated and expanded for 14 days in the presence of 50 ng/ml IL-15then cytotoxicity against K562 target cells was tested in the presence or absence of Pyk2 inhibitor PF431396. Freshly isolated, non-expanded NK cellswere used as a control and similarly treated with PF431396. (F) WT or CD56-KO NK92 cells were incubated for 7 days in the presence of 50 ng/ml IL-15then cytotoxicity was tested in the presence or absence of PF431396. Shown is one representative experiment from three independent biologicalrepeats. Error bars represent 3 technical repeats, SEM.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 15 of 16Research article Cell Biology Immunology and InflammationFigure 7. CD56 co-localizes with pPyk2 Y402 to the immune synapse in primary human NK cells. Primary NK cells were enriched from peripheral bloodthen incubated with K562 target cells for 45 min on poly-L-lysine coated coverslips in the presence of non-blocking anti-CD56 antibody. Followingincubation cells were fixed, permeabilized, and immunostained for pPyk2 Y402 (magenta), perforin (cyan) and actin (phalloidin, greyscale). 3Dvolumetric images were acquired by spinning disk confocal microscopy. (A) Representative images from one of three healthy donors. Shown is amaximum projection of 13 planes taken with 0.5 mm steps. (B) Fluorescence intensity of CD56 (left) or pPyk2 Y402 (right) measured at the synaptic (IS) ornon-synaptic (NK) cell cortex of primary NK cells conjugated to target cells. n = 22 from one independent experiment of 3 using three different healthydonors. **p<0.005, ****p<0.0001 by paired t-test.Gunesch et al. eLife 2020;9:e57346. DOI: https://doi.org/10.7554/eLife.57346 16 of 16Research article Cell Biology Immunology and Inflammation

CD56 regulates human NK cell cytotoxicity through Pyk2

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CD56 regulates human NK cell cytotoxicity through Pyk2

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