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Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy
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| Zeitschriftentitel: | Blood |
|---|---|
| Personen und Körperschaften: | , , , , , , , |
| In: | Blood, 134, 2019, Supplement_1, S. 1943-1943 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society of Hematology
|
| Schlagwörter: |
| author_facet |
Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang |
|---|---|
| author |
Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang |
| spellingShingle |
Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang Blood Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy Cell Biology Hematology Immunology Biochemistry |
| author_sort |
herda, stefanie |
| spelling |
Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood-2019-124736 <jats:p>Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture.</jats:p> <jats:p>Method:</jats:p> <jats:p>Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients.</jats:p> <jats:p>Results:</jats:p> <jats:p>There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice.</jats:p> <jats:p>Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually.</jats:p> <jats:p>Conclusion:</jats:p> <jats:p>Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.</jats:p> </jats:sec> Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy Blood |
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10.1182/blood-2019-124736 |
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Medizin Chemie und Pharmazie Biologie |
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American Society of Hematology, 2019 |
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American Society of Hematology, 2019 |
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0006-4971 1528-0020 |
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0006-4971 1528-0020 |
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American Society of Hematology |
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| title |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_unstemmed |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_full |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_fullStr |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_full_unstemmed |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_short |
Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_sort |
long-term t cell expansion results in increased numbers of central memory t cells with sustained functional properties for adoptive t cell therapy |
| topic |
Cell Biology Hematology Immunology Biochemistry |
| url |
http://dx.doi.org/10.1182/blood-2019-124736 |
| publishDate |
2019 |
| physical |
1943-1943 |
| description |
<jats:p>Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture.</jats:p>
<jats:p>Method:</jats:p>
<jats:p>Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients.</jats:p>
<jats:p>Results:</jats:p>
<jats:p>There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice.</jats:p>
<jats:p>Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually.</jats:p>
<jats:p>Conclusion:</jats:p>
<jats:p>Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts.</jats:p>
<jats:sec>
<jats:title>Disclosures</jats:title>
<jats:p>Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.</jats:p>
</jats:sec> |
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| author | Herda, Stefanie, Heimann, Andreas, Althoff, Stefanie, Ruß, Josefine, Bullinger, Lars, Beule, Dieter, Obermayer, Benedikt, Na, Il-Kang |
| author_facet | Herda, Stefanie, Heimann, Andreas, Althoff, Stefanie, Ruß, Josefine, Bullinger, Lars, Beule, Dieter, Obermayer, Benedikt, Na, Il-Kang, Herda, Stefanie, Heimann, Andreas, Althoff, Stefanie, Ruß, Josefine, Bullinger, Lars, Beule, Dieter, Obermayer, Benedikt, Na, Il-Kang |
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| description | <jats:p>Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture.</jats:p> <jats:p>Method:</jats:p> <jats:p>Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients.</jats:p> <jats:p>Results:</jats:p> <jats:p>There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice.</jats:p> <jats:p>Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually.</jats:p> <jats:p>Conclusion:</jats:p> <jats:p>Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.</jats:p> </jats:sec> |
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| imprint | American Society of Hematology, 2019 |
| imprint_str_mv | American Society of Hematology, 2019 |
| institution | DE-D275, DE-Bn3, DE-Brt1, DE-D161, DE-Zwi2, DE-Gla1, DE-Zi4, DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229 |
| issn | 0006-4971, 1528-0020 |
| issn_str_mv | 0006-4971, 1528-0020 |
| language | English |
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| match_str | herda2019longtermtcellexpansionresultsinincreasednumbersofcentralmemorytcellswithsustainedfunctionalpropertiesforadoptivetcelltherapy |
| mega_collection | American Society of Hematology (CrossRef) |
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| spelling | Herda, Stefanie Heimann, Andreas Althoff, Stefanie Ruß, Josefine Bullinger, Lars Beule, Dieter Obermayer, Benedikt Na, Il-Kang 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood-2019-124736 <jats:p>Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture.</jats:p> <jats:p>Method:</jats:p> <jats:p>Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients.</jats:p> <jats:p>Results:</jats:p> <jats:p>There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice.</jats:p> <jats:p>Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually.</jats:p> <jats:p>Conclusion:</jats:p> <jats:p>Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.</jats:p> </jats:sec> Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy Blood |
| spellingShingle | Herda, Stefanie, Heimann, Andreas, Althoff, Stefanie, Ruß, Josefine, Bullinger, Lars, Beule, Dieter, Obermayer, Benedikt, Na, Il-Kang, Blood, Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy, Cell Biology, Hematology, Immunology, Biochemistry |
| title | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_full | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_fullStr | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_full_unstemmed | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_short | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| title_sort | long-term t cell expansion results in increased numbers of central memory t cells with sustained functional properties for adoptive t cell therapy |
| title_unstemmed | Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy |
| topic | Cell Biology, Hematology, Immunology, Biochemistry |
| url | http://dx.doi.org/10.1182/blood-2019-124736 |