Generation and flow cytometric quality control of clinical‐scale <scp>TCRαβ/CD</scp>19‐depleted grafts

Bremm, Melanie; Cappel, Claudia; Erben, Stephanie; Jarisch, Andrea; Schumm, Michael; Arendt, Andreas; Bönig, Halvard; Klingebiel, Thomas; Koehl, Ulrike; Bader, Peter; Huenecke, Sabine

doi:10.1002/cyto.b.21328

Cited by 12 publications

(16 citation statements)

References 27 publications

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“…As the number of residual T cells in the graft might be a limiting factor regarding SC dose, an exact strategy to determine the graft constitution is essential. Therefore, we and others applied a single‐platform flow‐cytometric approach including 7‐AAD for viability analysis . After CD3/CD19 depletion, residual T and B cells are at least partly labeled with depletion antibodies, possibly leading to reduced fluorescence intensity within flow‐cytometric analysis.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we and others applied a single-platform flow-cytometric approach including 7-AAD for viability analysis. 16,30,31 After CD3/CD19 depletion, residual T and B cells are at least partly labeled with depletion antibodies, possibly leading to reduced fluorescence intensity within flowcytometric analysis. Therefore, for precise residual T-cell analysis, CD3 with SK7 clone, which binds to another epitope than the depletion antibody, was applied.…”

Section: Discussionmentioning

confidence: 99%

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Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation

et al. 2016

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation

et al. 2016

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“…Myeloablative protocols included myeloablative Bu/Flu (targeted Bu area under the curve, >70 mg*h/L) or Treo/Flu/thiotepa (TT). Graft manipulation strategies used were as follows: (1) CD34 1 selection 16 with add-back of 1 to 3 3 10 8 /kg CD3 1 T cells (CD34 1 / T-cell add-back graft), (2) TCRab and B-cell depletion, 17 (3) unmanipulated CB, and (4) unmanipulated BM or PBSCs. Details on selection of the conditioning regimen, graft manipulation strategy, and T-cell add-back dose among CD34 1 -selected grafts are provided in the Methods section in this article's Online Repository at www.jacionline.org.…”

Section: Donor Source Hla Typing Conditioning Protocol and Graft Mmentioning

confidence: 99%

New graft manipulation strategies improve the outcome of mismatched stem cell transplantation in children with primary immunodeficiencies

Elfeky

Shah

Unni

et al. 2019

Journal of Allergy and Clinical Immunology

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Background: Mismatched stem cell transplantation is associated with a high risk of graft loss, graft-versus-host disease (GvHD), and transplant-related mortality. Alternative graft manipulation strategies have been used over the last 11 years to reduce these risks. Objective: We investigated the outcome of using different graft manipulation strategies among children with primary immunodeficiencies. Methods: Between 2006 and 2017, 147 patients with primary immunodeficiencies received 155 mismatched grafts: 30 T-cell receptor (TCR) ab/CD19-depleted grafts, 43 cord blood (CB) grafts (72% with no serotherapy), 17 CD34 1 selection with Tcell add-back grafts, and 65 unmanipulated grafts. Results: The estimated 8-year survival of the entire cohort was 79%, transplant-related mortality was 21.7%, and the graft failure rate was 6.7%. Posttransplantation viral reactivation, grade II to IV acute graft-versus-host disease (aGvHD), and chronic graft-versus-host disease (cGvHD) complicated 49.6%, 35%, and 15% of transplantations, respectively. Use of TCRab/ CD19 depletion was associated with a significantly lower incidence of grade II to IV aGvHD (11.5%) and cGvHD (0%), although with a greater incidence of viral reactivation (70%) in comparison with other grafts. T-cell immune reconstitution was robust among CB transplants, although with a high incidence (56.7%) of grade II to IV aGvHD. Stable full donor engraftment was significantly greater at 80% among TCRab 1 /CD19 1-depleted and CB transplants versus 40% to 60% among the other groups. Conclusions: Rapidly accessible CB and haploidentical grafts are suitable alternatives for patients with no HLA-matched donor. Cord transplantation without serotherapy and TCRab 1 / CD19 1-depleted grafts produced comparable survival rates of around 80%, although with a high rate of aGvHD with the former and a high risk of viral reactivation with the latter that need to be addressed.

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“…An effective strategy to prevent the occurrence of lethal GvHD is the in vitro depletion of T cells. Specific depletion techniques are available to remove T and B cells from the grafts (CD3/CD19- or TCRαβ/CD19-depletion) while preserving cells that are beneficial regarding engraftment and GvL/T effect such as NK cells (9, 34, 35). Notably, HLA class I expression is reduced on NB cells which makes them valid targets for NK cell lysis (10, 36).…”

Section: Discussionmentioning

confidence: 99%

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

et al. 2019

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Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients.

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Generation and flow cytometric quality control of clinical‐scale TCRαβ/CD19‐depleted grafts

Cited by 12 publications

References 27 publications

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation

New graft manipulation strategies improve the outcome of mismatched stem cell transplantation in children with primary immunodeficiencies

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

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