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Monitoring Circulating γδ T Cells in Cancer Patients to Optimize γδ T Cell-Based Immunotherapy.

Oberg HH, Kellner C, Peipp M, Sebens S, Adam-Klages S, Gramatzki M, Kabelitz D, Wesch D - Front Immunol (2014)

Bottom Line: The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells.A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article.Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel , Kiel , Germany.

ABSTRACT
The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.

No MeSH data available.


Related in: MedlinePlus

Correlation between absolute cell number and functional capacity of Vδ2 T cells. (A) Flow cytometric analysis of CD3+ Vδ2+ γδTc within PBMC, and RTCA of PBMC from two representative donors (Donors 7 and 2) of 21; (B) list of the relative and absolute numbers (abs.) of CD3, γδ, Vδ2, and non-Vδ2 T cells in whole blood from 11 representative PDAC patients out of 21 as well as reactivity to the tribody; (C) flow cytometric analysis of selective expansion of CD3+ Vδ2+ γδTc after PAg-activation within PBMC for 8 days, and RTCA with these short-term expanded γδTc from Donors 7 and 2. Two representative donors of 21 are shown. (A,C) For RTCA, 5 × 103 PDAC cells (PancTu-I) were cultured in 10% FCS RPMI medium for 24–27 h on an E-plate covered at the bottom with electronic sensors that measure the impedance of the cells expressed as an arbitrary unit called cell index (CI). The CI was analyzed every 5 min to determine adherence and thus cell growth. Since the initial adherence in different wells can differ slightly, the CI was normalized to 1 shortly before the time of addition of suspended cells ± substances (vertical black line). After 24–27 h, PDAC cells were treated again with medium [green line (0)] or with PBMC (A) or short-term expanded γδTc (C) together with medium [orange line (i)], 300 nM PAg BrHPP [dark blue line (ii)], or 1 μg/ml [(Her2)2xVγ9)] [red line (iii)] at the indicated E:T ratio over the indicated time. As a positive control for maximal lysis, PDAC cells were treated with Triton X-100 [TX-100, black line (iv)]. The addition of substances, PBMC or expanded γδTc is indicated by the blue arrow. CI was then measured every minute for analysis of precise cytotoxicity time point for >15 to 55 h as indicated. The loss of tumor cell impedance and thus a decrease of the Normalized CI correlates with γδ T cell-mediated lysis of PDAC cells. The red arrow with the * points out the initiation of cytotoxicity. The average of triplicates and standard deviation were calculated; one representative experiment is shown.
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Figure 2: Correlation between absolute cell number and functional capacity of Vδ2 T cells. (A) Flow cytometric analysis of CD3+ Vδ2+ γδTc within PBMC, and RTCA of PBMC from two representative donors (Donors 7 and 2) of 21; (B) list of the relative and absolute numbers (abs.) of CD3, γδ, Vδ2, and non-Vδ2 T cells in whole blood from 11 representative PDAC patients out of 21 as well as reactivity to the tribody; (C) flow cytometric analysis of selective expansion of CD3+ Vδ2+ γδTc after PAg-activation within PBMC for 8 days, and RTCA with these short-term expanded γδTc from Donors 7 and 2. Two representative donors of 21 are shown. (A,C) For RTCA, 5 × 103 PDAC cells (PancTu-I) were cultured in 10% FCS RPMI medium for 24–27 h on an E-plate covered at the bottom with electronic sensors that measure the impedance of the cells expressed as an arbitrary unit called cell index (CI). The CI was analyzed every 5 min to determine adherence and thus cell growth. Since the initial adherence in different wells can differ slightly, the CI was normalized to 1 shortly before the time of addition of suspended cells ± substances (vertical black line). After 24–27 h, PDAC cells were treated again with medium [green line (0)] or with PBMC (A) or short-term expanded γδTc (C) together with medium [orange line (i)], 300 nM PAg BrHPP [dark blue line (ii)], or 1 μg/ml [(Her2)2xVγ9)] [red line (iii)] at the indicated E:T ratio over the indicated time. As a positive control for maximal lysis, PDAC cells were treated with Triton X-100 [TX-100, black line (iv)]. The addition of substances, PBMC or expanded γδTc is indicated by the blue arrow. CI was then measured every minute for analysis of precise cytotoxicity time point for >15 to 55 h as indicated. The loss of tumor cell impedance and thus a decrease of the Normalized CI correlates with γδ T cell-mediated lysis of PDAC cells. The red arrow with the * points out the initiation of cytotoxicity. The average of triplicates and standard deviation were calculated; one representative experiment is shown.

Mentions: To ensure adherence of tumor cells, PDAC cells were cultured for 24–27 h in RTCA plates before the addition of γδTc alone with or without additional substances. Thereafter, PDAC cells were still cultured alone or together with PDAC patient-derived PBMC in (i) medium, (ii) PAg such as bromohydrin-pyrophosphate (BrHPP), or (iii) [(Her2)2xVγ9]. During the extended time course, we observed that γδTc within PBMC required almost 24–36 h after initial stimulation to exert their cytotoxic capacity (Figure 2A, red arrow with a star). Moreover, we observed that [(Her2)2xVγ9] triggered tumor cell lysis more efficiently than PAg in 30% of PDAC patient samples (Figure 2A, responder), while neither substance was effective in 70% of patient samples (Figure 2A, non-responder). The unexpected cytotoxicity against PDAC cells in the absence of a stimulus (medium, orange line) is likely due to the reactivity of NK cells in the presence of IL-2 (Figure 2A), because additional experiments with untouched, freshly isolated γδTc demonstrated that cytotoxic activity of γδTc is not induced by IL-2 alone (16).


Monitoring Circulating γδ T Cells in Cancer Patients to Optimize γδ T Cell-Based Immunotherapy.

Oberg HH, Kellner C, Peipp M, Sebens S, Adam-Klages S, Gramatzki M, Kabelitz D, Wesch D - Front Immunol (2014)

Correlation between absolute cell number and functional capacity of Vδ2 T cells. (A) Flow cytometric analysis of CD3+ Vδ2+ γδTc within PBMC, and RTCA of PBMC from two representative donors (Donors 7 and 2) of 21; (B) list of the relative and absolute numbers (abs.) of CD3, γδ, Vδ2, and non-Vδ2 T cells in whole blood from 11 representative PDAC patients out of 21 as well as reactivity to the tribody; (C) flow cytometric analysis of selective expansion of CD3+ Vδ2+ γδTc after PAg-activation within PBMC for 8 days, and RTCA with these short-term expanded γδTc from Donors 7 and 2. Two representative donors of 21 are shown. (A,C) For RTCA, 5 × 103 PDAC cells (PancTu-I) were cultured in 10% FCS RPMI medium for 24–27 h on an E-plate covered at the bottom with electronic sensors that measure the impedance of the cells expressed as an arbitrary unit called cell index (CI). The CI was analyzed every 5 min to determine adherence and thus cell growth. Since the initial adherence in different wells can differ slightly, the CI was normalized to 1 shortly before the time of addition of suspended cells ± substances (vertical black line). After 24–27 h, PDAC cells were treated again with medium [green line (0)] or with PBMC (A) or short-term expanded γδTc (C) together with medium [orange line (i)], 300 nM PAg BrHPP [dark blue line (ii)], or 1 μg/ml [(Her2)2xVγ9)] [red line (iii)] at the indicated E:T ratio over the indicated time. As a positive control for maximal lysis, PDAC cells were treated with Triton X-100 [TX-100, black line (iv)]. The addition of substances, PBMC or expanded γδTc is indicated by the blue arrow. CI was then measured every minute for analysis of precise cytotoxicity time point for >15 to 55 h as indicated. The loss of tumor cell impedance and thus a decrease of the Normalized CI correlates with γδ T cell-mediated lysis of PDAC cells. The red arrow with the * points out the initiation of cytotoxicity. The average of triplicates and standard deviation were calculated; one representative experiment is shown.
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Related In: Results  -  Collection

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Figure 2: Correlation between absolute cell number and functional capacity of Vδ2 T cells. (A) Flow cytometric analysis of CD3+ Vδ2+ γδTc within PBMC, and RTCA of PBMC from two representative donors (Donors 7 and 2) of 21; (B) list of the relative and absolute numbers (abs.) of CD3, γδ, Vδ2, and non-Vδ2 T cells in whole blood from 11 representative PDAC patients out of 21 as well as reactivity to the tribody; (C) flow cytometric analysis of selective expansion of CD3+ Vδ2+ γδTc after PAg-activation within PBMC for 8 days, and RTCA with these short-term expanded γδTc from Donors 7 and 2. Two representative donors of 21 are shown. (A,C) For RTCA, 5 × 103 PDAC cells (PancTu-I) were cultured in 10% FCS RPMI medium for 24–27 h on an E-plate covered at the bottom with electronic sensors that measure the impedance of the cells expressed as an arbitrary unit called cell index (CI). The CI was analyzed every 5 min to determine adherence and thus cell growth. Since the initial adherence in different wells can differ slightly, the CI was normalized to 1 shortly before the time of addition of suspended cells ± substances (vertical black line). After 24–27 h, PDAC cells were treated again with medium [green line (0)] or with PBMC (A) or short-term expanded γδTc (C) together with medium [orange line (i)], 300 nM PAg BrHPP [dark blue line (ii)], or 1 μg/ml [(Her2)2xVγ9)] [red line (iii)] at the indicated E:T ratio over the indicated time. As a positive control for maximal lysis, PDAC cells were treated with Triton X-100 [TX-100, black line (iv)]. The addition of substances, PBMC or expanded γδTc is indicated by the blue arrow. CI was then measured every minute for analysis of precise cytotoxicity time point for >15 to 55 h as indicated. The loss of tumor cell impedance and thus a decrease of the Normalized CI correlates with γδ T cell-mediated lysis of PDAC cells. The red arrow with the * points out the initiation of cytotoxicity. The average of triplicates and standard deviation were calculated; one representative experiment is shown.
Mentions: To ensure adherence of tumor cells, PDAC cells were cultured for 24–27 h in RTCA plates before the addition of γδTc alone with or without additional substances. Thereafter, PDAC cells were still cultured alone or together with PDAC patient-derived PBMC in (i) medium, (ii) PAg such as bromohydrin-pyrophosphate (BrHPP), or (iii) [(Her2)2xVγ9]. During the extended time course, we observed that γδTc within PBMC required almost 24–36 h after initial stimulation to exert their cytotoxic capacity (Figure 2A, red arrow with a star). Moreover, we observed that [(Her2)2xVγ9] triggered tumor cell lysis more efficiently than PAg in 30% of PDAC patient samples (Figure 2A, responder), while neither substance was effective in 70% of patient samples (Figure 2A, non-responder). The unexpected cytotoxicity against PDAC cells in the absence of a stimulus (medium, orange line) is likely due to the reactivity of NK cells in the presence of IL-2 (Figure 2A), because additional experiments with untouched, freshly isolated γδTc demonstrated that cytotoxic activity of γδTc is not induced by IL-2 alone (16).

Bottom Line: The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells.A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article.Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel , Kiel , Germany.

ABSTRACT
The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.

No MeSH data available.


Related in: MedlinePlus