Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 19 (8), 2132-43

Growth and Activation of Natural Killer Cells Ex Vivo From Children With Neuroblastoma for Adoptive Cell Therapy

Affiliations

Growth and Activation of Natural Killer Cells Ex Vivo From Children With Neuroblastoma for Adoptive Cell Therapy

Yin Liu et al. Clin Cancer Res.

Abstract

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).

Experimental design: Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.

Results: Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56(+)CD3(-)CD14(-) NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151-9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.

Conclusion: Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Propagation of NK cells from PBMC of patient and normal donors by co-culture with gamma-irradiated K562 Clone 9.mbIL21 aAPC (total cells: aAPC ratio = 2:1) and 50 IU/mL IL-2 (A) Total cell growth curves for 5 normal donors and 10 neuroblastoma patients (see Materials and Methods for cell culture details; arrows indicate the days when aAPC were added to cultures). Recovery of viable cells after 7 and 14 days of co-culture with aAPC was determined by trypan blue exclusion. The slopes of the total viable cell growth curves for patients and healthy donors were identical (p=0.33). (B) and (C) Representative immunophenotyping results for a patient’s PBMC before (B) and after 14 days (C) co-culture with aAPC supplemented with 50 IU/mL IL-2. Aliquots of day 0 and 14 cells were viably frozen and then thawed for analysis on the same day. (D) Mean and standard deviation of immunophenotyping data from normal donors (n=5) and patients (n=10). CD19 was not included in the analysis of specimens from normal donors on days 0 and 14 (n.d., not done).
Figure 2
Figure 2
Expression of activation markers and receptors on NK cells (CD56medCD16+CD3CD14 and CD56highCD16CD3CD14) from neuroblastoma patients before and after 14 days of co-culture with aAPC plus 50 IU/mL IL-2. (A) Histogram overlays show expression of natural cytotoxicity receptors (DNAM-1, NKG2D, and NKp46), adhesion molecule (CD56), and FcγRIII receptor (CD16). Results are representative of experiments testing PBMCs from ten patients. CD3+CD14+and CD19+ cells were excluded from the analysis electronically. (B) Relative expression levels of markers and receptors on NK cells before and after 14 days of co-culture. Mean and standard deviation of ratios of mean fluorescence intensity (MFI) ratios of NK cells on day 14/day 0 from ten NBL patients are shown.
Figure 3
Figure 3
Anti-neuroblastoma cytotoxicity of effector cells expanded from PBMC after 14-day co-culture with K562 Clone 9.mbIL21 aAPC. Cytotoxicity of aNK alone (aNK cytotoxicity) or combined with anti-GD2 antibody ch14.18 (aNK ADCC) against neuroblastoma cell lines was determined using a 6-hour calcein-AM assay. aNK from 5 healthy donors (A) and 10 neuroblastoma patients (B) were tested at different effector:target (E:T) cell ratios against neuroblastoma cell lines LA-N-1, CHLA-255-Fluc and CHLA-136. Target cell survival (mean ± SD) at the indicated E:T ratios for all experiments are shown (6 replicate wells per condition per experiment). Linear regression was used to compare aNK cytotoxicity and aNK ADCC for the three tumor cell lines. Tumor cell survival for all cell lines was greater as the E:T ratio decreased (P<0.001) and was greater for aNK alone compared to aNK combined with ch14.18 (P=0.03, =0.009, and =0.007 for LA-N-1, CHLA-255-Fluc, and CHLA-136 respectively).
Figure 4
Figure 4
Cytokine and chemokine release from K562 Clone 9.mbIL21 aAPC-expanded aNK cells after a 24-hour incubation with neuroblastoma cells lines CHLA-255-Fluc and CHLA-136 alone or with anti-GD2 antibody ch14.18. Day 14 expanded aNK cells from 5 neuroblastoma patient donors were thawed and cultured for 72 hours with 50 IU/mL IL-2 prior to co-culture with CHLA255-Fluc or CHLA-136 cells (1:1 E:T ratio, 24 hours) without or with ch14.18, and then supernatants were collected for both Luminex® and the CBA assays. Means and standard deviation of fold changes are shown in dots and lines, respectively; red square dots indicate significant P values (P<0.05) for each comparison versus aNK cells alone. Fold changes and concentration (pg/ml) of cytokines or chemokines secreted by aNK cells exposed to different conditions (numerator in each function) are shown on the right-hand side of each data point.
Figure 5
Figure 5
Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells that were thawed and immediately infused or thawed, cultured, and then infused into NOD/SCID mice with disseminated neuroblastoma. .Cryopreserved effector cells (80% CD56+CD3CD14 NK) derived from normal donor PBMC were thawed and cultured for 3 days or thawed and immediately injected intravenously into NOD/SCID mice beginning 7 days after they received 106 CHLA-255-Fluc neuroblastoma cells intravenously. All mice receiving effector cells also received IL-2 (3 µg/mouse intravenously, 2x/week). Anti-GD2 mAb ch14.18 (15 µg/mouse intravenously, 2x/week) was given to the indicated groups. (A) Neuroblastoma tumor growth was visualized 34 and 48 days after tumor cell injection using bioluminescence imaging. Neuroblastoma progressed in all 8 untreated mice (control group) who died or were euthanized from days 38 to 43. (B) Signal intensities (total Flux) were detected at the time points shown in control and treated mice and plotted as mean ± SD. Comparison of Area Under the Curve (AUC) for the groups showed significant differences, with the exception of thawed and immediately injected aNK + ch14.18 versus thawed, cultured and injected aNK + ch14.18. (C) Overall survival curves for all treatment groups were generated by Kaplan-Meier analysis. Comparison of survival for the three groups showed significant difference, except for thawed and immediately injected aNK + ch14.18 versus thawed, cultured and injected aNK + ch14.18. Note that PBMC were isolated and cryopreserved at Children’s Hospital Los Angeles and shipped frozen to MD Anderson Cancer Center for NK cell growth, cryopreservation, and shipment back to Children’s Hospital for the experiment.
Figure 6
Figure 6
Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells when treatment was begun 7 days (early treatment) after tumor cell injection. Effector cells (77% CD56+CD3CD14 NK) derived from a normal donor PBMC after 21 days of culture were cryopreserved and then thawed and immediately injected through the tail vein into NOD/SCID mice that had received 106 CHLA-255-Fluc neuroblastoma cells intravenously 7 days previously. All mice receiving effector cells also received IL-2 (3 µg /mouse intravenously, 2x/week) with each aNK injection. Anti-GD2 mAb ch14.18 (15 µg/mouse intravenously, 2x/week) for each aNK injection was given to indicated groups. (A) Neuroblastoma cell growth was visualized 20 and 55 days after tumor cell injection using bioluminescence imaging. Neuroblastoma progressed in all 7 untreated control mice who died or were euthanized from days 35 to 42. (B) Signal intensities (total Flux) were detected at the time-points shown in control and treated mice and plotted as mean ± SD. Comparison of AUCs demonstrated that all treatments had an anti-tumor effect with aNK cells combined with ch14.18 having the greatest effect as shown in the see inset table. (C) Survival curves for all groups were generated by Kaplan-Meier analysis. All treatments significantly improved survival with aNK cells combined with ch14.18 being the most effective as shown in the inset table. The surviving mouse in this group did not have detectible disease at day 100 but did so at days 35 and 55.

Similar articles

See all similar articles

Cited by 33 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback