![Figure 1. CD56dimCD16− NK cell phenotype can be induced by cryopreservation. (A-B) Representative flow cytometry plots of fresh and cryopreserved NK cells from HSCT recipients at 4 weeks after transplantation (A) and healthy donors (B). (C-D) Longitudinal NK cell reconstitution in 9 HSCT recipients, based on the paired evaluation of fresh (C) and cryopreserved (D) PBMCs. Shown is the contribution (mean ± SEM) of CD56brightCD16+/− (light gray), CD56dimCD16+ (dark gray), and CD56dimCD16− (black) cells to the NK cell compartment. (E) Evaluation of fresh (left) and cryopreserved (right) NK cells after direct (<6 hours) or delayed (24 hours) separation of PBMCs from EDTA blood. Shown is the mean ± SEM of 4 healthy donors. (F-G) Contribution of early differentiated NKG2A+KIR− NK cells (F) and cryopreservation-induced CD56dimCD16− NK cells (G) to the total NK cell population, expressed as a function of time after HSCT. (H) Differentiation of NK cells based on the expression of NKG2A and KIRs on NK cells in fresh (F) and cryopreserved (C) PBMCs. Shown is the percentage of NKG2A+KIR− (light gray), NKG2A+KIR+ (white), NKG2A−KIR+ (dark gray), and NKG2A−KIR+ (black) NK cells divided over the CD56brightCD16+/− and CD56dimCD16+ NK cell phenotypes. Bars represent mean ± SEM of 4 healthy donors or 2 HSCT recipients at 4 weeks after transplantation. Flow cytometry-data were acquired on a Becton Dickinson LSRII flow cytometer and analyzed using Beckman Coulter (BC) Kaluza software. Gating strategy (A-E,G): forward scatter vs DAPI− (Sigma-Aldrich): live → CD45+ (FITC, clone 2D1, BD) vs CD14− (PE, MOP9, BD), CD33− (PE, P67.6, BD) and CD235a− (PE, KC16, BC) → CD19− (APC, J4.119, BC) → CD3− (Brilliant Violet 421, UCHT1, BD) vs CD7+ (Alexa 700, M-T701, BD) → CD56 (PE-Texas Red, N901, BC) vs CD16 (Brilliant Violet 711, 3G8, BD). Gating strategy (F,H): forward scatter vs DAPI− (Sigma-Aldrich): live → CD14− (APC, MOP9, BD) and CD19− (APC, J4.119, BC) → CD3− (Brilliant Violet 421, UCHT1, BD) vs CD7+ (Alexa 700, M-T701, BD) → CD56 (PE-Texas Red, N901, BC) vs CD16 (Brilliant Violet 711, 3G8, BD) → NKG2A (PE-Cy7, Z199, BC) vs KIR (PE, combination of CD158e + a/h + i + b/j [DX9, BD + EB6, BC + FES172, BC + GL183, BC]).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/125/11/10.1182_blood-2014-11-610311/4/1842f1.jpeg?Expires=1722581568&Signature=kZ5hrX2HvyyENNgl2fEhLJ1VZfOSe9UbXj8o-~hwuJ9GonLP1gZj~ziLPz6I0RynHO5kTaw3KPieQxoQe~8AiGxFiwdoRXiDGAowD8ar3x-w8WHM5jYGqm5kAQmGCkh1AhbDcluDcrC25s42atj7LJ64JcKlpH-NFSimPFglvN~Jl1vuFWJdxlHACv0kNSreEAi2wRbtvksO9GSiaaVJfP5-I0XX7wgKL1iCJpjllydQ~0rWi0zDZqZ9CE96nu0ULQasO8UXGOdWPcJFltnvo8JQmOuEQTHz-5i2U3Apfw1qChfnJWWKKVz4kQ70IQDg~tYDp3BQsIubrVxA2J~Z8g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD56dimCD16− NK cell phenotype can be induced by cryopreservation. (A-B) Representative flow cytometry plots of fresh and cryopreserved NK cells from HSCT recipients at 4 weeks after transplantation (A) and healthy donors (B). (C-D) Longitudinal NK cell reconstitution in 9 HSCT recipients, based on the paired evaluation of fresh (C) and cryopreserved (D) PBMCs. Shown is the contribution (mean ± SEM) of CD56brightCD16+/− (light gray), CD56dimCD16+ (dark gray), and CD56dimCD16− (black) cells to the NK cell compartment. (E) Evaluation of fresh (left) and cryopreserved (right) NK cells after direct (<6 hours) or delayed (24 hours) separation of PBMCs from EDTA blood. Shown is the mean ± SEM of 4 healthy donors. (F-G) Contribution of early differentiated NKG2A+KIR− NK cells (F) and cryopreservation-induced CD56dimCD16− NK cells (G) to the total NK cell population, expressed as a function of time after HSCT. (H) Differentiation of NK cells based on the expression of NKG2A and KIRs on NK cells in fresh (F) and cryopreserved (C) PBMCs. Shown is the percentage of NKG2A+KIR− (light gray), NKG2A+KIR+ (white), NKG2A−KIR+ (dark gray), and NKG2A−KIR+ (black) NK cells divided over the CD56brightCD16+/− and CD56dimCD16+ NK cell phenotypes. Bars represent mean ± SEM of 4 healthy donors or 2 HSCT recipients at 4 weeks after transplantation. Flow cytometry-data were acquired on a Becton Dickinson LSRII flow cytometer and analyzed using Beckman Coulter (BC) Kaluza software. Gating strategy (A-E,G): forward scatter vs DAPI− (Sigma-Aldrich): live → CD45+ (FITC, clone 2D1, BD) vs CD14− (PE, MOP9, BD), CD33− (PE, P67.6, BD) and CD235a− (PE, KC16, BC) → CD19− (APC, J4.119, BC) → CD3− (Brilliant Violet 421, UCHT1, BD) vs CD7+ (Alexa 700, M-T701, BD) → CD56 (PE-Texas Red, N901, BC) vs CD16 (Brilliant Violet 711, 3G8, BD). Gating strategy (F,H): forward scatter vs DAPI− (Sigma-Aldrich): live → CD14− (APC, MOP9, BD) and CD19− (APC, J4.119, BC) → CD3− (Brilliant Violet 421, UCHT1, BD) vs CD7+ (Alexa 700, M-T701, BD) → CD56 (PE-Texas Red, N901, BC) vs CD16 (Brilliant Violet 711, 3G8, BD) → NKG2A (PE-Cy7, Z199, BC) vs KIR (PE, combination of CD158e + a/h + i + b/j [DX9, BD + EB6, BC + FES172, BC + GL183, BC]).
