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Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

Cho FN, Chang TH, Shu CW, Ko MC, Liao SK, Wu KH, Yu MS, Lin SJ, Hong YC, Chen CH, Hung CH, Chang YH - PLoS ONE (2014)

Bottom Line: However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome.In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy.This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.

ABSTRACT
Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

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Related in: MedlinePlus

Immunophenotypic features of expanded NK cells (acceptor cells) and K562-antiCD19BBζ cells (feeder cells).A. Expression of CD56 and CD3 on peripheral blood mononuclear cells from a healthy donor was examined after 1 week (top row) of co-culture with irradiated (IR, left column) or freeze/thaw-treated (F, right column) K562-mb15-41BBL cells at a low dose (10 U/mL) of IL-2. The T cells were removed using CD3 Dynabeads, generating cell populations comprising >95% CD56+CD3- NK cells (bottom row). B. Percentage of CD56-positive cells within NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells prior to and after CD3 depletion on day 7. The data are presented as the mean of values obtained using 3 unrelated NK donors. Error bars represent the SD. C. Histogram illustrating the anti-CD19 expression on K562 cells (control, shaded histogram) and K562-antiCD19BBζ cells (feeder cells, open histogram).
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pone-0109352-g001: Immunophenotypic features of expanded NK cells (acceptor cells) and K562-antiCD19BBζ cells (feeder cells).A. Expression of CD56 and CD3 on peripheral blood mononuclear cells from a healthy donor was examined after 1 week (top row) of co-culture with irradiated (IR, left column) or freeze/thaw-treated (F, right column) K562-mb15-41BBL cells at a low dose (10 U/mL) of IL-2. The T cells were removed using CD3 Dynabeads, generating cell populations comprising >95% CD56+CD3- NK cells (bottom row). B. Percentage of CD56-positive cells within NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells prior to and after CD3 depletion on day 7. The data are presented as the mean of values obtained using 3 unrelated NK donors. Error bars represent the SD. C. Histogram illustrating the anti-CD19 expression on K562 cells (control, shaded histogram) and K562-antiCD19BBζ cells (feeder cells, open histogram).

Mentions: The NK cells were expanded from PBMCs by co-culturing with irradiated (Figure 1A, left panels) or freeze/thaw-treated (Figure 1A, right panels) K562-mb15-41BBL cells. The freeze/thaw cycle compromised the membrane integrity of K562-mb15-41BBL cells, which allowed trypan blue staining, but intact cell morphology was maintained. After 7 days of expansion, the PBMCs co-cultured with irradiated K562-mbIL15-41BBL cells produced 95.8% CD56+CD3- NK cells (Figure 1A, left upper panel), whereas K562-mbIL15-41BBL cells subjected to one freeze/thaw cycle yielded 81.8% CD56+CD3- cells (Figure 1A, right upper panel). After CD3 depletion, the percentages of CD56+CD3- NK cells were 98.7% from PBMCs co-cultured with irradiated K562-mbIL15-41BBL cells and 95.5% using a freeze/thawed procedure (Figure 1A, lower panels). The relatively poor expansion of NK cells in co-cultures of PMBCs and freeze/thaw-treated K562-mbIL15-41BBL cells was likely due to the reduced stimulation from mbIL15 and 41BB ligands as a result of freeze/thaw-induced cell damage and lysis.


Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

Cho FN, Chang TH, Shu CW, Ko MC, Liao SK, Wu KH, Yu MS, Lin SJ, Hong YC, Chen CH, Hung CH, Chang YH - PLoS ONE (2014)

Immunophenotypic features of expanded NK cells (acceptor cells) and K562-antiCD19BBζ cells (feeder cells).A. Expression of CD56 and CD3 on peripheral blood mononuclear cells from a healthy donor was examined after 1 week (top row) of co-culture with irradiated (IR, left column) or freeze/thaw-treated (F, right column) K562-mb15-41BBL cells at a low dose (10 U/mL) of IL-2. The T cells were removed using CD3 Dynabeads, generating cell populations comprising >95% CD56+CD3- NK cells (bottom row). B. Percentage of CD56-positive cells within NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells prior to and after CD3 depletion on day 7. The data are presented as the mean of values obtained using 3 unrelated NK donors. Error bars represent the SD. C. Histogram illustrating the anti-CD19 expression on K562 cells (control, shaded histogram) and K562-antiCD19BBζ cells (feeder cells, open histogram).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196898&req=5

pone-0109352-g001: Immunophenotypic features of expanded NK cells (acceptor cells) and K562-antiCD19BBζ cells (feeder cells).A. Expression of CD56 and CD3 on peripheral blood mononuclear cells from a healthy donor was examined after 1 week (top row) of co-culture with irradiated (IR, left column) or freeze/thaw-treated (F, right column) K562-mb15-41BBL cells at a low dose (10 U/mL) of IL-2. The T cells were removed using CD3 Dynabeads, generating cell populations comprising >95% CD56+CD3- NK cells (bottom row). B. Percentage of CD56-positive cells within NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells prior to and after CD3 depletion on day 7. The data are presented as the mean of values obtained using 3 unrelated NK donors. Error bars represent the SD. C. Histogram illustrating the anti-CD19 expression on K562 cells (control, shaded histogram) and K562-antiCD19BBζ cells (feeder cells, open histogram).
Mentions: The NK cells were expanded from PBMCs by co-culturing with irradiated (Figure 1A, left panels) or freeze/thaw-treated (Figure 1A, right panels) K562-mb15-41BBL cells. The freeze/thaw cycle compromised the membrane integrity of K562-mb15-41BBL cells, which allowed trypan blue staining, but intact cell morphology was maintained. After 7 days of expansion, the PBMCs co-cultured with irradiated K562-mbIL15-41BBL cells produced 95.8% CD56+CD3- NK cells (Figure 1A, left upper panel), whereas K562-mbIL15-41BBL cells subjected to one freeze/thaw cycle yielded 81.8% CD56+CD3- cells (Figure 1A, right upper panel). After CD3 depletion, the percentages of CD56+CD3- NK cells were 98.7% from PBMCs co-cultured with irradiated K562-mbIL15-41BBL cells and 95.5% using a freeze/thawed procedure (Figure 1A, lower panels). The relatively poor expansion of NK cells in co-cultures of PMBCs and freeze/thaw-treated K562-mbIL15-41BBL cells was likely due to the reduced stimulation from mbIL15 and 41BB ligands as a result of freeze/thaw-induced cell damage and lysis.

Bottom Line: However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome.In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy.This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.

ABSTRACT
Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

Show MeSH
Related in: MedlinePlus