Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients’ Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays
Abstract
:1. Introduction
1.1. Current Therapy Strategies for Acute Myeloid Leukemia (AML)
1.2. Dendritic Cells for Immune Therapy
1.3. Innate and Adaptive Immune Systems
1.4. Intracellular Cytokine Assay (INCYT), Degranulation Assay (DEG), and Cytotoxicity Fluorolysis Assay (CTX)
1.5. Aims of This Study
2. Results
2.1. Prologue
2.2. Effects of LAA-/SEB-Stimulation in Uncultured (D0) WB (AML and Healthy) on the Intracellular IFNy-Production and Degranulation (CD107a) Positivity of Immune Cell Subtypes
2.3. Effects of Kit M on the Generation of DC/DCleu and Blast Proliferation Using AML and Healthy WB
2.4. Effects of Kit M-Treated vs. Untreated AML WB before and after T Cell-Enriched MLC on Immunoreactive Cells
2.5. Composition of IFNy- and TNFa-Producing Immune-Reactive Cells after T Cell-Enriched MLC, Using Leukemic WB with and without (Control) Kit-Pretreatment (Kit M) and with (+LAA) and without (-LAA) LAA-Stimulation
2.6. Effects of Kit M-Treated vs. Untreated AML WB on the Degranulation of Immune Cells after MLC
2.7. Effects of Kit M-Treated WB on the Antileukemic and Blastolytic Activity after MLC, Detected via CTX
2.8. Correlation of the Relative Improvement of IFNy and TNFa Secretion and the Degranulation with the Relative Improvement of Blast Lysis in Kit M-Treated (vs. Untreated) WB after MLC
2.9. Cases with Intracellular IFNy Production before and after MLC, Subdivided into Cytogenetic ELN Risk Groups (Favorable and Adverse)
2.10. Cases with Intracellular IFNy Production before and after MLC, Subdivided into Patients’ Response to Induction Chemotherapy
3. Discussion
3.1. Use of DC-Based Immunotherapy in AML
3.2. Generation of DCs Ex Vivo
3.3. Different Age Groups in AML Patients’ WB Samples and Healthy Donors
3.4. Detection of Leukemia-Specific Cells
3.5. Leukemia-Specific Activity of Immune-Reactive Cells
3.6. Correlation of Results of CTX Values and Leukemia-Specific Assays such as INCYT (INFy and TNFa) and DEG
4. Material and Methods
4.1. Sample Acquisition
4.2. Patients’ Characteristics
4.3. Sample Preparation
4.4. Characterization and Quantification of Cells via Flow Cytometry
4.5. Dendritic Cell Culture (DCC)
4.6. Mixed Lymphocyte Culture (MLC)
4.7. Degranulation Assay (DEG) and Intracellular Cytokine Assay (INCYT)
4.8. Intracellular Cytokine Assay (INTCYT)
4.9. Cytotoxicity Fluorolysis Assay (CTX)
4.10. Statistical Methods
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Type | Name of Subgroups | Abbreviation of Subgroups | Surface Marker | Referred to | Abbreviation | Reference |
---|---|---|---|---|---|---|
Subtypes of blasts and DC | ||||||
Blast cells | Leukemic blasts | Bla | Bla+ (e.g., CD34+, CD177+) | WB | Bla/WB | [26] |
Proliferating blasts | Blaprol-CD71 | Bla+DC−CD71+ | Bla | Blaprol-CD71/Bla | [26] | |
Proliferating blasts | Blaprol-IPO38 | Bla+DC−IPO38+ | Bla | Blaprol-IPO38/Bla | [26] | |
Dendritic cells | Dendritic cells | DC | DC+ (CD80+, CD206+) | WB | DC/cells | [26] |
Leukemia derived DC | DCleu | DC+Bla+ | WB | DCleu/cells | [26] | |
Bla | DCleu/Bla | [26] | ||||
Mature DC | DCmig | DC+CD197+ | WB | DCmig/cells | [26] | |
Mature DCleu | DCleu-mig | DC+CD197+Bla+ | WB | DCleu-mig/cells | [26] | |
DC | DCleu-mig/DC | [26] | ||||
DCleu | DCleu-mig/DCleu | [26] | ||||
Bla | DCleu-mig/Bla | [26] | ||||
Subtypes of immune-reactive cells | ||||||
T cells | CD3+ pan T cells | T | CD3+ | WB | T/cells | [10] |
CD4+ coexpressing T cells | T4+ | CD3+CD4+ | T | T4+/T | [10] | |
CD8+ coexpressing T cells | T4- | CD3+CD8+ | T | T4-/T | [10] | |
Proliferating T cells-early | Tprol-early | CD3+CD69+ | T | Tprol-early/T | [10] | |
Proliferating T cells-late | Tprol-late | CD3+CD71+ | T | Tprol-late/T | [10] | |
Non-naive T cells | Tnon-naive | CD3+CD45RO+ | T | Tnon-naive/T | [8] | |
Central (memory) T cells | Tcm | CD3+CD45RO+CD197+ | T | Tcm/T | [8] | |
Subtypes of different degranulating (CD107a+) cells | ||||||
T cells | CD3+ pan T cells | T107a | CD107a+CD3+ | T | T107a/T | [27] |
CD4+ coexpressing T cells | T4+107a | CD107a+CD3+CD4+ | T | T4+107a/T | [10] | |
CD8+ coexpressing T cells | T4-107a | CD107a+CD3+CD8+ | T | T4-107a/T | [10] | |
Non-naive T cells | Tnon-naive107a | CD107a+CD3+CD45RO+ | Tnon-naive | Tnon-naive107a/Tnon-naive | [27] | |
Effector (memory) T cells | Tem107a | CD107a+CD3+CD45RO+CD197− | Tem | Tem107a/Tem | [27] | |
Central (memory) T cells | Tcm107a | CD107a+CD3+CD45RO+CD197+ | Tcm | Tcm107a/Tcm | [27] | |
NK cells | CD3−CD56+ NK cells | NK107a | CD107a+CD3−CD56+ | NK | NK107a/NK | [27] |
CIK cells | CD3+CD56+ CIK cells | CIK107a | CD107a+CD3+CD56+ | CIK | CIK107a/CIK | [27] |
B cells | CD19+ | B107a | CD107a+CD19+ | B | B107a/B | [27] |
Subtypes of different intracellularly IFNy or TNFa-producing cells | ||||||
T cells | CD3+ pan T cells | TIFNy/TNFa | IFNy+/TNFa+CD3+ | T | TIFNy/TNFa/T | [8] |
CD4+-coexpressing T cells | T4+IFNy/TNFa | IFNy+/TNFa+CD3+ CD4+ | T4+ | T4+IFNy/TNFa/T4+ | [8] | |
CD8+-coexpressing T cells | T4-IFNy/TNFa | IFNy+/TNFa+CD3+CD8+ | T4- | T4-IFNy/TNFa/T4- | [8] | |
Non-naive T cells | Tnon-naive IFNy/TNFa | IFNy+/TNFa+CD3+CD45RO+ | Tnon-naive | Tnon-naiveIFNy/TNFa/Tnon-naive | [8] | |
Effector (memory) T cells | Tem IFNy/TNFa/Tem | IFNy+/TNFa+CD3+CD45RO+CD197− | Tem | Tem IFNy/TNFa/Tem | [8] | |
Central (memory) T cells | Tcm IFNy/TNFa | IFNy+/TNFa+CD3+CD45RO+CD197+ | Tcm | Tcm IFNy/TNFa/Tcm | [8] | |
NK cells | CD3−CD56+ NK cells | NKIFNy/TNFa | IFNy+/TNFa+CD3−CD56+ | NK | NKIFNy/TNFa/NK | [8] |
CIK cells | CD3+CD56+ CIK cells | CIKIFNy/TNFa | IFNy+/TNFa+CD3+CD56+ | CIK | CIKIFNy/TNFa/CIK | [8] |
Patient No. | Sex | Age at dgn | FAB type | Stage | Blast in PB (%) * | Blast Phenotype (CD) | ELN-Risk- Stratification | Response to Induction Chemotherapy | Conducted Experiments |
---|---|---|---|---|---|---|---|---|---|
AML | |||||||||
P1509 | m | 60 | pAML/M2 | dgn | 48 | 13,33,34,65,117 | favorable | response | DCC, MLC, INCYT, CSA, CTX |
P1514 | m | 68 | SAML | dgn | 51 | 33,56,117 | favorable | no response | DCC, MLC, INCYT, DEG |
P1518 | f | 83 | pAML/M5 | dgn | 72 | 14,15,34,65 | favorable | no response | DCC, MLC, INCYT, CSA |
P1526 | f | 74 | pAML | dgn | 61 | 15,33,34,56,65,117 | favorable | n.d. | DCC, MLC, DEG, CSA |
P1602 | m | 67 | pAML | dgn | 16 | 13,33,34,56,117 | favorable | response | DCC, MLC, CTX |
P1630 | m | 29 | pAML | dgn | 16 | 13,15,33,34,64,117 | Tavorable | response | DCC, MLC, INCYT, DEG, CTX |
P1512 | f | 80 | pAML | dgn | 40 | 13,34,117 | adverse | no response | DCC, MLC, INCYT, DEG |
P1567 | f | 98 | SAML | dgn | 16 | 15,34,65,117 | adverse | no response | DCC, MLC, INCYT, DEG, CTX |
P1572 | f | 63 | SAML | dgn | 12 | 13,33,34,65,117 | adverse | response | DCC, MLC, INCYT, DEG, CTX |
P1573 | m | 61 | pAML/M6 | dgn | 13 | 13,34,65,71,117 | adverse | no response | DCC, MLC, INCYT, DEG, CTX |
P1581 | m | 56 | pAML/M4 | dgn | 58 | 13,15,33,34,117 | adverse | no response | DCC, MLC, INCYT, DEG, CTX |
P1638 | m | 68 | sAML | dgn | 33 | 4,13,33,34,56,117 | adverse | no response | DCC, MLC, INCYT, DEG, CTX |
P1527 | m | 42 | pAML/M2 | dgn | 28 | 7,13,15,33,34,65,117 | intermediate | n.d. | DCC, MLC, INCYT, DEG, CSA, CTX |
P1604 | f | 60 | sAML/MDS | dgn | 16 | 7,13,33,34,65,117 | intermediate | n.d. | DCC, MLC, INCYT, DEG, CTX |
P1635 | m | 51 | pAML | dgn | 25 | 15,33,34,117 | intermediate | response | DCC, MLC, INCYT, DEG |
P1594 | f | 70 | pAML/M4 | Per. disease | 20 | 13,33,34,65,117 | DCC, MLC, INCYT, DEG, CTX | ||
P1595 | f | 50 | pAML | Per. disease | 15 | 13,33,34,56,65,117 | DCC, MLC, CTX | ||
P1597 | f | 83 | sAML | Per. disease | 54 | 15,33,34,56,65,117 | DCC, MLC, INCYT, DEG, CTX | ||
P1603 | f | 32 | pAML | Per. disease | 50 | 15,33,34,117 | DCC, MLC, INCYT, DEG, CTX | ||
P1616 | f | 69 | pAML | Per. disease | 16 | 33,34,117 | DCC, MLR, INCYT, DEG | ||
P1497(2) | m | 74 | SAML | relapse | 59 | 13,34,65,117 | DCC, MLR, INCYT, DEG, CTX | ||
P1516 | f | 52 | SAML | relapse | 70 | 15,33,34,65,117 | DCC, MLR, INCYT, DEG, CTX | ||
P1522 | m | 47 | SAML | relapse | 55 | 13,34,71,117 | DCC, MLR, INCYT, DEG | ||
P1598 | f | 61 | sAML | relapse | 25 | 13,33,34,117 | DCC, MLR, INCYT, DEG, CTX | ||
P1599 | f | 71 | pAML/M4 | relapse | 79 | 7,13,33,34,117 | DCC, MLC, DEG, CTX | ||
P1632 | f | 56 | pAML | relapse | 65 | 13,33,34,56,65,117 | DCC, MLC, INCYT, DEG, CTX | ||
HEALTHY | |||||||||
P1510 | m | 22 | DCC, MLC, INCYT, DEG | ||||||
P1513 | m | 27 | DCC, MLC, INCYT, DEG | ||||||
P1517 | m | 39 | DCC, MLC, INCYT, DEG | ||||||
P1523 | m | 17 | DCC, MLC, INCYT, DEG, CSA | ||||||
P1566 | f | 54 | DCC, MLC, INCYT, DEG | ||||||
P1579 | m | 30 | DCC, MLC, INCYT, DEG | ||||||
P1580 | f | 24 | DCC, MLC, INCYT, DEG | ||||||
P1582 | m | 27 | DCC, MLC, INCYT, DEG | ||||||
P1583 | m | 28 | DCC, MLC, INCYT, DEG | ||||||
P1585 | m | 29 | DCC, MLC, INCYT, DEG | ||||||
P1586 | m | 29 | DCC, MLC | ||||||
P1590 | f | 23 | DCC, MLC | ||||||
P1592 | f | 58 | DCC, MLC, INCYT, DEG | ||||||
P1596 | m | 26 | DCC, MLC | ||||||
P1611 | f | 27 | DCC, MLC, INCYT, DEG | ||||||
P1613 | f | 24 | DCC, MLC, INCYT, DEG | ||||||
P1636 | m | 22 | DCC, MLC, INCYT, DEG | ||||||
P1637 | f | 22 | DCC, MLC, INCYT, DEG |
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Schutti, O.; Klauer, L.; Baudrexler, T.; Burkert, F.; Schmohl, J.; Hentrich, M.; Bojko, P.; Kraemer, D.; Rank, A.; Schmid, C.; et al. Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients’ Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays. Int. J. Mol. Sci. 2024, 25, 6983. https://doi.org/10.3390/ijms25136983
Schutti O, Klauer L, Baudrexler T, Burkert F, Schmohl J, Hentrich M, Bojko P, Kraemer D, Rank A, Schmid C, et al. Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients’ Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays. International Journal of Molecular Sciences. 2024; 25(13):6983. https://doi.org/10.3390/ijms25136983
Chicago/Turabian StyleSchutti, Olga, Lara Klauer, Tobias Baudrexler, Florian Burkert, Joerg Schmohl, Marcus Hentrich, Peter Bojko, Doris Kraemer, Andreas Rank, Christoph Schmid, and et al. 2024. "Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients’ Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays" International Journal of Molecular Sciences 25, no. 13: 6983. https://doi.org/10.3390/ijms25136983