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NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2.

Braciak TA, Wildenhain S, Roskopf CC, Schubert IA, Fey GH, Jacob U, Hopfner KP, Oduncu FS - J Transl Med (2013)

Bottom Line: NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity.The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstrasse 1, D-80336 Munich, Germany. Todd.Braciak@med.uni-muenchen.de.

ABSTRACT

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.

Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays.

Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission.

Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

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Unaltered expression patterns of the NCRs NKp30, NKp44 and NKp46 on enriched NKs from the patient at diagnosis, in remission and the healthy twin. (A) Expression pattern of NCRs on enriched NKs from the patient in remission (red) and the healthy twin (blue) compared with cells stained with an isotype control mAb (black). (B) To analyze NCR expression on enriched NKs from the patient at diagnosis, an additional sorting step for CD16 positive cells was performed to focus the analysis on the small subpopulation of NKs. NKs from the patient at diagnosis (red) are compared with cells stained with an isotype control mAb (black). (C) Gates chosen to generate data shown in panels A) and B). Left: gate chosen to identify MACS-sorted cells (negative selection for NK cells). Right: additional gate used for identification of the CD16 pos subset (lower right quadrant) of the MACS sorted cells from the patient sample obtained at diagnosis (Methods).
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Figure 6: Unaltered expression patterns of the NCRs NKp30, NKp44 and NKp46 on enriched NKs from the patient at diagnosis, in remission and the healthy twin. (A) Expression pattern of NCRs on enriched NKs from the patient in remission (red) and the healthy twin (blue) compared with cells stained with an isotype control mAb (black). (B) To analyze NCR expression on enriched NKs from the patient at diagnosis, an additional sorting step for CD16 positive cells was performed to focus the analysis on the small subpopulation of NKs. NKs from the patient at diagnosis (red) are compared with cells stained with an isotype control mAb (black). (C) Gates chosen to generate data shown in panels A) and B). Left: gate chosen to identify MACS-sorted cells (negative selection for NK cells). Right: additional gate used for identification of the CD16 pos subset (lower right quadrant) of the MACS sorted cells from the patient sample obtained at diagnosis (Methods).

Mentions: As impaired cytolytic activity of NKs from AML patients had been proposed to be caused by reduced expression of NCRs [25,26], expression of NKp30, NKp44 and NKp46 on enriched NKs was studied (Figure 6). No significant difference in the expression patterns was observed between MACS enriched NKs from the patient in remission versus the healthy sibling (Figure 6A). The corresponding analysis for NKs from the patient at diagnosis was rendered more difficult by the fact that NKs were of low abundance in the MACS-enriched sample at this time. This analysis was therefore refined by further gating for CD16pos cells and then analyzing the NCRs on this gated population of NKs. In this manner, an essentially unchanged NCR expression profile was found for these gated NKs (Figure 6B). The gating strategy used for this analysis of the PBMC sample drawn at diagnosis is shown in Figure 6C. From these combined observations, we conclude that for this particular patient the NCR expression profiles remained unchanged and that changes in the levels of NCRs are unlikely to be the cause of the impaired cytolytic functions recorded here.


NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2.

Braciak TA, Wildenhain S, Roskopf CC, Schubert IA, Fey GH, Jacob U, Hopfner KP, Oduncu FS - J Transl Med (2013)

Unaltered expression patterns of the NCRs NKp30, NKp44 and NKp46 on enriched NKs from the patient at diagnosis, in remission and the healthy twin. (A) Expression pattern of NCRs on enriched NKs from the patient in remission (red) and the healthy twin (blue) compared with cells stained with an isotype control mAb (black). (B) To analyze NCR expression on enriched NKs from the patient at diagnosis, an additional sorting step for CD16 positive cells was performed to focus the analysis on the small subpopulation of NKs. NKs from the patient at diagnosis (red) are compared with cells stained with an isotype control mAb (black). (C) Gates chosen to generate data shown in panels A) and B). Left: gate chosen to identify MACS-sorted cells (negative selection for NK cells). Right: additional gate used for identification of the CD16 pos subset (lower right quadrant) of the MACS sorted cells from the patient sample obtained at diagnosis (Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842817&req=5

Figure 6: Unaltered expression patterns of the NCRs NKp30, NKp44 and NKp46 on enriched NKs from the patient at diagnosis, in remission and the healthy twin. (A) Expression pattern of NCRs on enriched NKs from the patient in remission (red) and the healthy twin (blue) compared with cells stained with an isotype control mAb (black). (B) To analyze NCR expression on enriched NKs from the patient at diagnosis, an additional sorting step for CD16 positive cells was performed to focus the analysis on the small subpopulation of NKs. NKs from the patient at diagnosis (red) are compared with cells stained with an isotype control mAb (black). (C) Gates chosen to generate data shown in panels A) and B). Left: gate chosen to identify MACS-sorted cells (negative selection for NK cells). Right: additional gate used for identification of the CD16 pos subset (lower right quadrant) of the MACS sorted cells from the patient sample obtained at diagnosis (Methods).
Mentions: As impaired cytolytic activity of NKs from AML patients had been proposed to be caused by reduced expression of NCRs [25,26], expression of NKp30, NKp44 and NKp46 on enriched NKs was studied (Figure 6). No significant difference in the expression patterns was observed between MACS enriched NKs from the patient in remission versus the healthy sibling (Figure 6A). The corresponding analysis for NKs from the patient at diagnosis was rendered more difficult by the fact that NKs were of low abundance in the MACS-enriched sample at this time. This analysis was therefore refined by further gating for CD16pos cells and then analyzing the NCRs on this gated population of NKs. In this manner, an essentially unchanged NCR expression profile was found for these gated NKs (Figure 6B). The gating strategy used for this analysis of the PBMC sample drawn at diagnosis is shown in Figure 6C. From these combined observations, we conclude that for this particular patient the NCR expression profiles remained unchanged and that changes in the levels of NCRs are unlikely to be the cause of the impaired cytolytic functions recorded here.

Bottom Line: NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity.The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstrasse 1, D-80336 Munich, Germany. Todd.Braciak@med.uni-muenchen.de.

ABSTRACT

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared.

Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays.

Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission.

Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

Show MeSH
Related in: MedlinePlus