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Defining novel parameters for the optimal priming and expansion of minor histocompatibility antigen-specific T cells in culture.

Janelle V, Carli C, Taillefer J, Orio J, Delisle JS - J Transl Med (2015)

Bottom Line: Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases.While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression.Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche de l'Hôpital Maisonneuve-Rosemont, Montreal, Quebec, Canada. vjanelle@pcitp.org.

ABSTRACT

Background: Adoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion.

Methods: Using the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of "fit" MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays.

Results: Following a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure.

Conclusion: Our results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

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Related in: MedlinePlus

Generation of MiHA-specific T-cell lines in coculture. (A) Kinetics of culture growth as represented by the fold increase in cell count from the number of PBMCs seeded at the beginning of the coculture step; n = 4. (B) left: one representative of the CD8+ HA-1+ cells generated over time analyzed by flow cytometry; middle: combination of 4 donors, data show average of donors ± SEM; right: variation of CD8+ HA-1+ over time for each donor. ns (non significant).
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Fig1: Generation of MiHA-specific T-cell lines in coculture. (A) Kinetics of culture growth as represented by the fold increase in cell count from the number of PBMCs seeded at the beginning of the coculture step; n = 4. (B) left: one representative of the CD8+ HA-1+ cells generated over time analyzed by flow cytometry; middle: combination of 4 donors, data show average of donors ± SEM; right: variation of CD8+ HA-1+ over time for each donor. ns (non significant).

Mentions: We monitored this coculture for a total of four weeks. According to our conditions, T-cell lines entered their exponential growth phase around day 14 and reached their maximal growth rate between days 21 and 28 to achieve a total of ~25 to 140-fold expansion (Figure 1A). In parallel, cocultures with unpulsed DCs were performed as control and showed more modest expansions, although the difference was not statistically significant.Figure 1


Defining novel parameters for the optimal priming and expansion of minor histocompatibility antigen-specific T cells in culture.

Janelle V, Carli C, Taillefer J, Orio J, Delisle JS - J Transl Med (2015)

Generation of MiHA-specific T-cell lines in coculture. (A) Kinetics of culture growth as represented by the fold increase in cell count from the number of PBMCs seeded at the beginning of the coculture step; n = 4. (B) left: one representative of the CD8+ HA-1+ cells generated over time analyzed by flow cytometry; middle: combination of 4 donors, data show average of donors ± SEM; right: variation of CD8+ HA-1+ over time for each donor. ns (non significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4413989&req=5

Fig1: Generation of MiHA-specific T-cell lines in coculture. (A) Kinetics of culture growth as represented by the fold increase in cell count from the number of PBMCs seeded at the beginning of the coculture step; n = 4. (B) left: one representative of the CD8+ HA-1+ cells generated over time analyzed by flow cytometry; middle: combination of 4 donors, data show average of donors ± SEM; right: variation of CD8+ HA-1+ over time for each donor. ns (non significant).
Mentions: We monitored this coculture for a total of four weeks. According to our conditions, T-cell lines entered their exponential growth phase around day 14 and reached their maximal growth rate between days 21 and 28 to achieve a total of ~25 to 140-fold expansion (Figure 1A). In parallel, cocultures with unpulsed DCs were performed as control and showed more modest expansions, although the difference was not statistically significant.Figure 1

Bottom Line: Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases.While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression.Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche de l'Hôpital Maisonneuve-Rosemont, Montreal, Quebec, Canada. vjanelle@pcitp.org.

ABSTRACT

Background: Adoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion.

Methods: Using the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of "fit" MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays.

Results: Following a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure.

Conclusion: Our results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

Show MeSH
Related in: MedlinePlus