Impact of various culture conditions on ex vivo expansion of polyclonal T cells for adoptive immunotherapy

Apmis ◽  
2019 ◽  
Vol 127 (12) ◽  
pp. 737-745 ◽  
Author(s):  
Sasan Ghaffari ◽  
Monireh Torabi‐Rahvar ◽  
Azadeh Omidkhoda ◽  
Naser Ahmadbeigi
Keyword(s):  
T Cells ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Ex Vivo Expansion

Ex Vivo Expansion of Umbilical Cord Blood T Cells for Adoptive Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 35-35
Author(s):  
Melissa A. Mazur ◽  
Young Ah Lee ◽  
Kurtzberg Joanne ◽  
Szabolcs Paul
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Cell Activity ◽  
Ex Vivo Expansion ◽  
Hla Dr

Abstract Background: Viral infections cause significant morbidity & mortality in patients undergoing unrelated allogeneic BMT transplantation before immune reconstitution is completed. It poses a greater risk for recipients of unrelated umbilical cord blood (uUCB) transplants as there is no established antiviral immunity in naïve UCB lymphocytes available for adoptive transfer. UCB T cells also lack Th1/Tc1 cytokines, Granzymes & Perforin which are prerequisites to control viral pathogens. Another major limitation of uUCBT is the lack of donor cells available for post-transplant donor leukocyte infusions (DLI) to boost immunity or induce GVL. However, a fraction of the uCB graft could be available for T cell expansion. In this study we evaluated the feasibility of ex vivo expansion of UCB T cells. We postulated that following expansion naïve T cells may mature & acquire a phenotype compatible with effector function as assessed by expression of essential cytokines & de novo expression of members of the granzyme-perforin pathway. Methods: Thawed UBC research samples with a leukocyte content <5% of an average UCB graft are processed. T cells are enriched with “EasySep” (StemCell Tech) to deplete CD14, CD16, CD19, CD56, & glycophorin A + cells. 5–7.5*105 T cells/ml are incubated with “CD3/28 T cell Expander” artificial APC beads (Dynal) in X Vivo-15 (BioWhittacker) + 200u/ml IL2 & 10% human serum in gas permeable bags. The initial purity of the T cells is 77–92%. The starting absolute T cell numbers ranged from 0.75 to 2*106 cells. Media & cytokines are added every other day to maintain a concentration of <2*106 cells/ml. Results: At the end of 14 days UCB T Cells expanded 67 fold +/− 36, n=6. There are significant alterations in phenotype over the 2 weeks (Table 1) with up to 40% of T cells in cell cycle. Compared to the starting resting UCB T cells the majority of expanded cells have acquired the phenotype of activated (HLA-DR+, CD25+ T cells) memory cells, at the expense of naive/recent thymic immigrants (CD45RA+/CD62+). There is an inverted CD4/CD8 ratio due to the higher expansion rate of CD8 T cells (p=0.0035) while there is no difference in apoptosis (p=0.57). However, they all retain expression of CD28 (96% ±8%) along with CD27. Although some T cells have acquired the capacity to secrete granzymes A and B these are still almost a log below normal adult peripheral blood (PB) values & perforin has not been detected. Similarly, while post expansion significantly more T cells secrete cytokines upon PMA + ionomycin stimulation (Table 1) they are below levels of adult PB. Conclusions: From our preliminary results we can demonstrate effective expansion & partial maturation of UCB T Cells. For example, if one starts with 2*106 total T cells & expands them 67 fold this could provide for DLI ~5*106 T cells/kg for an average pediatric patient (25kg). We are further optimizing & characterizing this model for T cell activity & repertoire. In sum, ex vivo expansion with CD3/CD28 co-stimulation may provide clinically relevant numbers T cells available for adoptive immunotherapy that have also undergone partial maturation. Characterization of Expanded T Cells as % of all Lymphocytes Variable Median SD CD3+ 99.8 0.1 CD4+ 35 11 CD4+/CD8+ 2.3 2.8 CD45RA+/RO− 13 11 CD45RO+/RA− 55 22 CD25+ 42 21 CD45RA+/CD62+ 38 20 CD45RA+/CD27+/CD8+ 52 15 CD45RA−/CD27+/CD8+ 46 15 KI67/CD8+ 42 9 Ki67/CD4+ 32 7 HLA DR+ 40 13 Granzyme A/CD8+ 54 18 Granzyme B/CD8+ 2 2 Perforin/CD8+ 0 0

Zoledronate facilitates large-scale ex vivo expansion of functional γδ T cells from cancer patients for use in adoptive immunotherapy*

Cytotherapy ◽  
2008 ◽  
Vol 10 (8) ◽  
pp. 842-856 ◽  
Author(s):  
M. Kondo ◽  
K. Sakuta ◽  
A. Noguchi ◽  
N. Ariyoshi ◽  
K. Sato ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Patients ◽  
Adoptive Immunotherapy ◽  
Large Scale ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Ex Vivo Expansion

scholarly journals Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement

2015 ◽  
Vol 4 (1) ◽  
pp. e31 ◽  
Author(s):  
Corey Smith ◽  
Grethe Økern ◽  
Sweera Rehan ◽  
Leone Beagley ◽  
Sau K Lee ◽  
...  
Keyword(s):  
T Cells ◽  
Adoptive Immunotherapy ◽  
Immune Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
The Novel ◽  
Serum Replacement ◽  
Human T Cells

Optimization of methods for ex vivo expansion of T-cells for adoptive immunotherapy

Cytotherapy ◽  
2017 ◽  
Vol 19 (5) ◽  
pp. S36
Author(s):  
M. Grau-Vorster ◽  
A. del Mazo-Barbara ◽  
D. Vivas-Pradillo ◽  
C. Mirabel ◽  
B. Reyes ◽  
...  
Keyword(s):  
T Cells ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Ex Vivo Expansion

Optimizing Culture Conditions for Ex Vivo Expansion of Virus Specific T Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4863-4863
Author(s):  
Gabriel Borelli ◽  
Tanja Aarvak ◽  
Anne Brunsvig ◽  
Marianne Dyrhaug ◽  
Marianne Lundby ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Ex Vivo ◽  
Treg Cells ◽  
Culture Conditions ◽  
Ex Vivo Expansion ◽  
T Cell Subsets ◽  
Clinical Grade

Abstract Adoptive immunotherapy with virus specific CD8+ cytotoxic T lymphocytes (CTL) is currently an option for treatment of viral infections after allogeneic stem cell transplantation. A major obstacle for clinical grade production of virus specific CTL is to retain the antigen specific clones, which can be easily deleted during ex vivo expansion. Manipulation of CD3/CD28 engagement, depletion of T regulatory (Treg) cells before expansion and prestimulation with antigen presenting cells were explored in this study. Mononuclear cells were obtained from HLA-A2+ donors by leucapheresis. Lymphocytes were enriched by elutriation, stimulated with anti-CD3/anti-CD28high Dynabeads or Dynabeads® ClinExVivo™ CD3/CD28 and cultured for 10 days in CellGro media supplemented with human AB serum and IL-2. Virus specific CD8+ CTL were quantified by flow using pentamer staining. By using anti-CD3/ anti-CD28high Dynabeads a more balanced expansion of CD4+ and CD8+ subsets was obtained, while Dynabeads ClinExVivo CD3/CD28 allowed a higher expansion of CD8+ subset and therefore a higher expansion of the virus specific CTL. A high Dynabead: T cell ratio (3:1) deleted completely the virus specific CTL. By reducing this ratio we could retain the virus specific CTL after expansion. No benefits were observed by adding extra Dynabeads during expansion. It is known that Treg cells can inhibit expansion of all T cell subsets. By depleting Treg cells with CD25 Dynabeads prior to T cell expansion, we observed a significantly increased expansion of all T cells, including virus specific CTL. In some patients low numbers of virus specific CTL can be detected. To increase the numbers of antigen specific CTL we have included a prestimulation step using peptide-loaded mononuclear cells prior to expansion with Dynabeads, which gave more than 3000- fold expansion of virus specific CTL. We are currently exploring the phenotype profile of expanded CTL (CD62L, CCR7, CD57, CD27, CD28 and CTLA-4), functionality (cytokine secretion and proliferative capacity) and cytotoxicity. This protocol can be upgraded to clinical grade production of virus specific CTL for treatment of viral infections in immunocompromised patients.

scholarly journals Closed loop bioreactor system for the ex vivo expansion of human T cells

Cytotherapy ◽  
2019 ◽  
Vol 21 (1) ◽  
pp. 76-82
Author(s):  
MATTHEW LI ◽  
LING-YEE CHIN ◽  
SYUKRI SHUKOR ◽  
ALFRED TAMAYO ◽  
MARCELA V. MAUS ◽  
...  
Keyword(s):  
T Cells ◽  
Closed Loop ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human T Cells ◽  
Bioreactor System

scholarly journals In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy

2021 ◽  
Vol 11 ◽  
Author(s):  
João Calmeiro ◽  
Luís Mendes ◽  
Iola F. Duarte ◽  
Catarina Leitão ◽  
Adriana R. Tavares ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Clinical Grade ◽  
Serum Free ◽  
Depth Analysis ◽  
Free Media ◽  
The Impact

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

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