Limits...
Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients’ plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs). (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE+/SytoxBlue−) and dead (CFSE+/SytoxBlue+) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t-test (D). ns, non-significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385630&req=5

Figure 4: Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs). (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE+/SytoxBlue−) and dead (CFSE+/SytoxBlue+) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t-test (D). ns, non-significant.

Mentions: To investigate the impact of sNKG2DLs on NK cell cytotoxicity, tumor spheroids were cocultured for 48 h at an E:T ratio of 5:1 with NK cells pre-incubated with a cocktail of sNKG2DLs (CAL27 SN: 27.63 ng/ml sNKG2DLs) or sNKG2DLs-depleted CAL27 supernatant (sNKG2DLs: 2.66 ng/ml) (Figures 4A,B). NK cells in basal culture medium served as controls. NK cells efficiently destroyed tumor spheroids (Figure 4A; Figure S4C in Supplementary Material). By contrast, in the presence of high levels of sNKG2DLs, NKG2D-dependent killing was reduced by 25% for FaDu, 19% for CAL27, and 46% for SiHa tumor spheroids (Figure 4A). Importantly, impaired tumor spheroid destruction and NKG2D-downregulation could be completely restored by specific depletion of sNKG2DLs (Figure 4A; Figure S4D in Supplementary Material).


Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma
Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs). (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE+/SytoxBlue−) and dead (CFSE+/SytoxBlue+) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t-test (D). ns, non-significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385630&req=5

Figure 4: Natural killer (NK) cell cytotoxicity and infiltration toward tumor spheroids is inhibited by shed NKG2DLs (sNKG2DLs). (A) NK cells incubated with non-/sNKG2DL-depleted CAL27 supernatant (SN) or medium (kill CO) prior to co-incubation with FaDu, CAL27, or SiHa tumor spheroids (E:T ratio 5:1, 48 h). Tumor spheroid lysis was calculated by analysis of live (CFSE+/SytoxBlue−) and dead (CFSE+/SytoxBlue+) tumor cells. Bars correspond to mean ± SEM of eight tumor spheroids. (B) sNKG2DL levels of non-/depleted CAL27 SN analyzed by ELISA. Data are shown as mean ± SEM of duplicates. (C) NK cells treated with CAL27 SN, sMICA01, or untreated (kill CO) prior to coculturing with FaDu cells for 48 h. Representative phase-contrast pictures at 50× magnification are shown (size bar = 100 µm). Cryosections of spheroids were stained for NK cells (anti-CD45 antibody, red) and apoptosis (anti-active caspase-3 antibody, brown). Representative pictures are shown at 200× magnification (size bar = 200 µm). (D) Percentage of infiltrated NK cells in FaDu, CAL27, or SiHa tumor spheroids. Bars correspond to mean ± SEM of 10 vision fields. Statistical significance was assessed by one-way analysis of variance (A) and unpaired, two-tailed Student’s t-test (D). ns, non-significant.
Mentions: To investigate the impact of sNKG2DLs on NK cell cytotoxicity, tumor spheroids were cocultured for 48 h at an E:T ratio of 5:1 with NK cells pre-incubated with a cocktail of sNKG2DLs (CAL27 SN: 27.63 ng/ml sNKG2DLs) or sNKG2DLs-depleted CAL27 supernatant (sNKG2DLs: 2.66 ng/ml) (Figures 4A,B). NK cells in basal culture medium served as controls. NK cells efficiently destroyed tumor spheroids (Figure 4A; Figure S4C in Supplementary Material). By contrast, in the presence of high levels of sNKG2DLs, NKG2D-dependent killing was reduced by 25% for FaDu, 19% for CAL27, and 46% for SiHa tumor spheroids (Figure 4A). Importantly, impaired tumor spheroid destruction and NKG2D-downregulation could be completely restored by specific depletion of sNKG2DLs (Figure 4A; Figure S4D in Supplementary Material).

View Article: PubMed Central - PubMed

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients’ plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus