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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

Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2–5 μg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45+ cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3+ (CD3), CD3+ TCRγδ+ (γδ), TCRγδ+ TCRnon-Vδ2+ (non-Vδ2), and TCRγδ+ TCRVδ2+ (Vδ2) within CD45+ lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 μl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.
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Figure 1: Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2–5 μg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45+ cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3+ (CD3), CD3+ TCRγδ+ (γδ), TCRγδ+ TCRnon-Vδ2+ (non-Vδ2), and TCRγδ+ TCRVδ2+ (Vδ2) within CD45+ lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 μl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.

Mentions: The monitoring system that uses the BD Multitest 6-color TBNK (M6T) Reagent with BD Trucount™ Beads (http://www.bd.com/resource.aspx?IDX=17743, BD Biosciences, San Jose, CA, US) allows determination of absolute cell numbers of αβ T and B lymphocytes and NK cells as well as CD4+ and CD8+ T cell subsets (17, 18). Since γδ T lymphocytes and their subpopulations are not detected by the M6T, we adapted γδTc staining from the BD Trucount™ Tube technical data sheet (version 8/2010) as follows: 50 μl whole blood from cancer patients were stained with anti-CD45-PE/Cy7 (clone HI30), CD3-PE (clone SK7) pan-TCRγδ-APC (clone 11F2, customized) (all from BD Biosciences, Heidelberg, Germany), and Vδ2-PerCP (clone B6, Biolegend, Fell, Germany) mAbs and occasionally with Vδ1-FITC mAb (clone TS8.2, Thermo Fisher Scientific, Germany) in BD Trucount™ Tubes as described (16). After staining, red blood cells were lysed with 200 μl BD Lysing buffer and analyzed using the FACS Canto flow cytometer and FACS Diva software (both from BD Biosciences). For two representative donors, the absolute numbers of total γδTc as well as Vδ2 and non-Vδ2 subsets are shown (Figure 1). Moreover, cells can be stained with anti-Vδ1 mAb labeled with an additional fluorochrome (data not shown).


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)

Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2–5 μg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45+ cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3+ (CD3), CD3+ TCRγδ+ (γδ), TCRγδ+ TCRnon-Vδ2+ (non-Vδ2), and TCRγδ+ TCRVδ2+ (Vδ2) within CD45+ lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 μl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269191&req=5

Figure 1: Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2–5 μg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45+ cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3+ (CD3), CD3+ TCRγδ+ (γδ), TCRγδ+ TCRnon-Vδ2+ (non-Vδ2), and TCRγδ+ TCRVδ2+ (Vδ2) within CD45+ lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 μl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.
Mentions: The monitoring system that uses the BD Multitest 6-color TBNK (M6T) Reagent with BD Trucount™ Beads (http://www.bd.com/resource.aspx?IDX=17743, BD Biosciences, San Jose, CA, US) allows determination of absolute cell numbers of αβ T and B lymphocytes and NK cells as well as CD4+ and CD8+ T cell subsets (17, 18). Since γδ T lymphocytes and their subpopulations are not detected by the M6T, we adapted γδTc staining from the BD Trucount™ Tube technical data sheet (version 8/2010) as follows: 50 μl whole blood from cancer patients were stained with anti-CD45-PE/Cy7 (clone HI30), CD3-PE (clone SK7) pan-TCRγδ-APC (clone 11F2, customized) (all from BD Biosciences, Heidelberg, Germany), and Vδ2-PerCP (clone B6, Biolegend, Fell, Germany) mAbs and occasionally with Vδ1-FITC mAb (clone TS8.2, Thermo Fisher Scientific, Germany) in BD Trucount™ Tubes as described (16). After staining, red blood cells were lysed with 200 μl BD Lysing buffer and analyzed using the FACS Canto flow cytometer and FACS Diva software (both from BD Biosciences). For two representative donors, the absolute numbers of total γδTc as well as Vδ2 and non-Vδ2 subsets are shown (Figure 1). Moreover, cells can be stained with anti-Vδ1 mAb labeled with an additional fluorochrome (data not shown).

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