A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus‐specific human CD4 and CD8 T lymphocytes

Pira, Giuseppina Li; Bottone, Laura; Ivaldi, Federico; Pelizzoli, Roberta; Risso, Marco; Tripodi, Gino; Manca, Fabrizio

doi:10.1111/j.1537-2995.2006.00977.x

Cited by 4 publications

(7 citation statements)

References 41 publications

(70 reference statements)

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“…An additional advantage of the reported system is that aliquots of autologous PBMCs separated in the sealed system can be frozen as a GMP product and used as APCs for later restimulation cycles. 18 We are now working on repeated stimulation cycles of T-cell lines to produce large numbers of specific T cells that can be frozen in multiple aliquots. It should also be noted that in our current experience, remarkable T-cell expansions on the basis of restimulation cycles can be obtained with leukopacks without the need of lymphoapheretic procedures (manuscript in preparation).…”

Section: Discussionmentioning

confidence: 99%

“…The CliniMacs apparatus (Miltenyi) has been used to select CMV-specific T cells in a sealed system, 13 Therefore, we adapted the sealed procedures we previously described 18 for PBMC separation, stimulation, selection, and final expansion of specific T cells. We used 10 CMV seropositive, HLA-A2 positive donors previously selected for their response to CMV antigens and peptides.…”

Section: Functional Assays On Expanded T-cell Lines Obtained From Thementioning

confidence: 99%

“…PBMCs were obtained by lymphocyte separation medium separation in sealed bags. 18 After washing and counting, the cells were resuspended in medium at 10 Â 10 6 /mL. Four milliliters of this suspension was transferred into a 25-mL Teflon bag prefilled with 4-mL peptide library prepared as a 2 Â solution in medium to obtain a final concentration of 2 ug/mL for each peptide.…”

Section: Pbmcs Preparation Selection and Processingmentioning

confidence: 99%

“…Blood was drawn in acid-citrate dextrose containing plastic bags when processed in the sealed system. All manipulations were performed as reported in detail 18 by keeping the sealed system rigorously unbreached. Additives, antigens, and media were prepared in advance in sealed bags (or tubings for volumes <2 mL) prefilled under sterile conditions.…”

Section: Pbmcs Preparation Selection and Processingmentioning

confidence: 99%

“…The different steps of the sealed procedure for separation of antigen-specific T cells on the basis of the cytokine capture method are represented in the flow diagram. A, PBMCs obtained as described in Ref 18. were stimulated with the peptide pool and incubated; if available, PBMC aliquots were frozen at this stage for later restimulation cycles.…”

mentioning

confidence: 99%

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Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems

Pira¹,

Ivaldi

Tripodi

et al. 2008

Journal of Immunotherapy

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Administration of pathogen-specific T-cell lines can reconstitute the cellular immune function of immunocompromised patients. Selection and expansion of specific T cells for reinfusion pose unique challenges owing to the fact that good manufacturing procedures must be implemented. Cytokine secretion-based methods can identify and select specific T cells. We showed here that it is possible to combine this method with procedures for cell handling performed in a sealed, unbreached system from start to end. Peripheral blood mononuclear cells, obtained from blood samples and processed in a sealed system, were stimulated in Teflon bags with a library of selected CD4 and CD8 peptides derived from the immunodominant cytomegalovirus protein pp65. The stimulated T cells were labeled with reagents for interferon-gamma surface capture and selected on a magnetic column using a sealed system connected to the Teflon bags. Elution and final expansion were also performed with an unbreached protocol with preservation of sterility even if the steps were run on the bench top. Expanded cells exhibited the appropriate functions. The use of this unbreached procedure proves that safety of cellular products generated in a good manufacturing procedures facility can be further improved. Similar sealed protocols can also be applied for T-cell therapies directed against tumor antigens.

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Section: Discussionmentioning

confidence: 99%

Section: Functional Assays On Expanded T-cell Lines Obtained From Thementioning

confidence: 99%

Section: Pbmcs Preparation Selection and Processingmentioning

confidence: 99%

Section: Pbmcs Preparation Selection and Processingmentioning

confidence: 99%

mentioning

confidence: 99%

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Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems

Pira¹,

Ivaldi

Tripodi

et al. 2008

Journal of Immunotherapy

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Selective Depletion of αβ T Cells and B Cells for Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation. A Three-Year Follow-Up of Procedure Efficiency

Pira

Malaspina

Girolami

et al. 2016

Biology of Blood and Marrow Transplantation

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HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αβ T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34 progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34 hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony-stimulating factor (G-CSF). Poor CD34 cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 10 nucleated cells and 494 × 10 CD34 HSC were manipulated using the CliniMACS device. In comparison to the initial product, αβ T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 10 CD34 HSC/kg recipient body weight, with median values of residual αβ T cells and B cells of 29 × 10/kg and 33 × 10/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34 cells was observed after the first year (P = .0005). These data indicate that αβ T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible.

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Recent developments in processing systems for cell and tissue cultures toward therapeutic application

Kino‐oka¹,

Taya²

2009

Journal of Bioscience and Bioengineering

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A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus‐specific human CD4 and CD8 T lymphocytes

Abstract: This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.

Cited by 4 publications

References 41 publications

Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems

Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems

Selective Depletion of αβ T Cells and B Cells for Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation. A Three-Year Follow-Up of Procedure Efficiency

Recent developments in processing systems for cell and tissue cultures toward therapeutic application

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