Skip to main page content (VSports最新版本)
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil VSports app下载. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2014 Nov 15;20(22):5708-19.
doi: 10.1158/1078-0432.CCR-13-3451. Epub 2014 May 15.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies

Drew C Deniger  1 , Sourindra N Maiti  2 , Tiejuan Mi  2 , Kirsten C Switzer  2 , Vijaya Ramachandran  3 , Lenka V Hurton  1 , Sonny Ang  2 , Simon Olivares  2 , Brian A Rabinovich  2 , M Helen Huls  2 , Dean A Lee  1 , Robert C Bast Jr  4 , Richard E Champlin  5 , Laurence J N Cooper  6
Affiliations

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies

"VSports手机版" Drew C Deniger et al. Clin Cancer Res. .

V体育官网 - Abstract

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. VSports手机版.

Experimental design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing V体育安卓版. .

Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. V体育ios版.

Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans VSports最新版本. .

PubMed Disclaimer

Figures

Figure 1
Figure 1. Sustained proliferation of polyclonal PBMC-derived γδT cells on γ-irradiated aAPC in presence of soluble IL-2 and IL-21
(A) Frequency of γδT cells before (Day 0) and after (Day 22) co-culture on γ-irradiated aAPC, IL-2, and IL-21 where expression of CD3, CD56, TCRαβ, TCRγδ, TCRδ1, and TCRδ2 is shown at Day 22 of co-culture. One of 7 representative donors is shown. Quadrant frequencies (percentage) within flow plots are displayed in upper right corners. (B) Inferred cell counts of polyclonal γδT cells are displayed calculated based on weekly yields and relative fold changes, where three arrows represent addition of aAPC. Black line is mean ± SD (n=4) pooled from 2 independent experiments and each gray line is an individual donor. (C) Fold increase over 9 days of γδT cells co-cultured with IL-2 and IL-21 along with aAPC expressing membrane-bound IL-15 (mIL15), CD86, and/or CD137L. Data are mean ± SD (n=3) pooled from 2 independent experiments and each shape represents an individual donor. Two-way ANOVA with Bonferroni’s post-tests was used for statistical analysis. *p<0.05 and **p<0.01 (D) Fold increase over 9 days of γδT cells co-cultured with aAPC (clone #4) in the presence of either soluble recombinant IL-2 and/or IL-21. Data are mean ± SD (n=3) pooled from 2 independent experiments where each shape represents an individual donor. Two-way ANOVA with Bonferroni’s post-tests was used for statistical analysis. *p<0.05
Figure 2
Figure 2. Abundance of Vδ and Vγ mRNA species in γδT cells propagated and activated ex vivo
Quantification of mRNA species coding for (A) Vδ and (B) Vγ alleles in PBMC-derived γδT cells by DTEA at day 22 of co-culture on aAPC/IL-2/IL-21. Quantification of mRNA species coding for (C) Vδ and (D) Vγ alleles in UCB-derived γδT cells by DTEA at day 34-35 of co-culture on aAPC/IL-2/IL-21. Box-and-whiskers plots display 25% and 75% percentiles where lines represent maximum, mean, and minimum from top to bottom (n=4). Solid lines at bottom of graphs represent limit-of-detection (LOD) calculated from mean ± 2xSD of DTEA negative controls. Student’s paired one-tailed t-tests were performed for each allele relative to the sample LOD. *p<0.05 and **p<0.01
Figure 3
Figure 3. Sustained proliferation of PBMC-derived Vδ T-cell subsets expanded on γ-irradiated aAPC/IL-2/IL-21
After two 7-day stimulations with aAPC (clone #4) and IL-2/IL-21 the bulk population of γδT cells were separated into Vδ1, Vδ2, and Vδ1negVδ2neg subsets by FACS based on staining of T cells defined as TCRδ1+TCRδ2neg, TCRδ1negTCRδ2+, and TCRδ1negTCRδ2neg, respectively. (A) Expression of TCRδ1 and TCRδ2 chains on Vδ1, Vδ2, and Vδ1negVδ2neg subsets of γδT cells (from left to right) after 15 days of numeric expansion on aAPC and cytokines as isolated groups. One of 4 representative donors is shown pooled from 2 independent experiments. Quadrant frequencies (percentage) within flow plots are displayed in upper right corners. Frequency of TCRδ1+TCRδ2neg (open bars), TCRδ1negTCRδ2+ (black bars), and TCRδ1negTCRδ2neg (gray bars) cell surface protein expression in subsets of γδT cells after 15 days numeric expansion on aAPC and cytokines as isolated groups. Data are mean ± SD (n=4) pooled from 2 independent experiments. (B) Flow cytometry plots of CD3 and TCRγδexpression in Vδ1, Vδ2, and Vδ1negVδneg subsets (from left to right). Mean fluorescence intensity (MFI) of TCRγδstaining in Vδ1, Vδ2, and Vδ1negVδneg T-cell subsets where each shape represents a different donor and data are mean ± SD (n=4) pooled from 2 independent experiments. (C) Proliferation of each isolated Vδ subset stimulated twice with aAPC clone #4 (arrows) in presence of cytokines and total cell counts are displayed. Data are mean ± SD (n=4) pooled from 2 independent experiments. (D) DTEA was used to identify and measure abundance of mRNA species coding for Vδ1*01, Vδ2*02, and Vδ3*01 (from left to right) in γδT-cell sub-populations after 15 days of proliferation on aAPC and cytokines as separated subsets. Box-and-whiskers plots display 25% and 75% percentiles where lines represent maximum, mean, and minimum from top to bottom (n=4). Student’s paired, two-tailed t-tests were undertaken for statistical analyses between groups. **p<0.01 and ***p<0.001
Figure 4
Figure 4. Dependence on TCRγδ for IFNγ secretion in response to tumor cells
At Day 22 of co-culture on γ-irradiated aAPC (clone #4) with IL-2 and IL-21, T cells were incubated with CM (mock) or leukocyte activation cocktail (LAC; PMA/Ionomycin) for 6 hours at 37 °C. Tissue culture supernatants were interrogated using 27-Plex Luminex array to detect presence of (A) TH1, TH2, and TH17 cytokines and selected chemokines (from left to right). Data are mean ± SD pooled from 4 donors in 2 independent experiments where each donor had triplicate experimental wells pooled prior to multiplex analysis. Student’s one-tailed t-test performed for statistical analysis between mock and LAC groups. *p<0.05, **p<0.01, and ***p<0.001 (B) Polyclonal γδT cells were incubated for 1 hour prior to and during 6 hour tumor cell co-culture with normal mouse serum or neutralizing TCRγδantibody (clone IM). Cells were stained for TCRδ1, TCRδ2, CD3, and IFNγ to gate T-cell subsets and assess IFNγ production. Comparisons of histograms detailing Vδ1, Vδ2, and Vδ1negVδneg gates (from left to right) co-cultured with CAOV3 ovarian cancer cells and treated with serum (open) or TCRγδ(shaded). Numbers next to histograms are MFI. Flow plots are representative of 1 of 3 PB donors co-cultured with CAOV3 cells in 2 independent experiments. (C) Percent inhibition of IFNγ secretion in response to CAOV3 cells was calculated for each Vδ T-cell subset based on the following equation: Inhibition (%) = 100 − 100 × [(MFITUMOR + TCELL − MFIT CELL ONLY)TCRγδ/(MFITUMOR + T CELL − MFIT CELL ONLY)Serum]. Data are mean ± SD (n=3) pooled from 2 independent experiments.
Figure 5
Figure 5. Specific lysis of tumor-cell panel by polyclonal γδT cells
(A-C) Standard 4-hour CRA was performed with increasing effector (polyclonal γδT cells; each shape represents a different donor) to target (E:T) ratios against (A) healthy B cells from an allogeneic donor (one of four representative donors), (B) hematological tumor cell lines derived from B-ALL: RCH-ACV, T-ALL: Jurkat, and CML: K562, (C) solid tumor cell lines derived from pancreatic cancer: BxPc-3, colon cancer: HCT-116, and ovarian cancer: OC314 and CAOV3. Data are mean ± SD (n=3 wells per assay) from 2 independent experiments. (D) Neutralizing antibodies to NKG2D (squares), DNAM1 (triangles), TCRγδ(inverted triangles), or a pool (diamonds) of all three antibodies were used to block killing of Jurkat (left), IGROV1 (middle), or OC314 (right) tumor targets antibodies at 0.3, 1, and 3 μg/mL and an E:T ratio of 12:1 in standard 4-hour CRA. Normal mouse serum (circles) served as control for addition of antibody and wells without antibody were used for normalization purposes. Specific lysis was normalized to wells without antibody to yield relative cytolysis as defined by: Relative cytolysis (%) = (Specific Lysis)With Antibody/(Specific Lysis)Without Antibody × 100. Data are mean ± SD (n=4 donors) from triplicates pooled and normalized from 2 independent experiments. Repeated-measures Two-way ANOVA was used for statistical analysis between antibody treatments. **p<0.01 and ***p<0.001
Figure 6
Figure 6. In vivo clearance of ovarian cancer upon adoptive transfer of polyclonal γδT cells and γδT-cell subsets propagated/activated on aAPC with IL-2 and IL-21
CAOV3-effLuc-mKate tumor cells were injected into NSG mice at Day -8 and engrafted until Day 0 when treatment was started with either PBS (vehicle/mock) or γδT cells. Four T-cell doses were administered in weekly escalating doses. (A) BLI images at Day 0 (top panels) or Day 72 (bottom panels) in PBS, Vδ1, Vδ2, Vδ1negVδneg, and polyclonal γδT-cell treatment groups. Images are representative of 6-14 mice from 2 independent experiments. (B) BLI measurements of mice at Day 0 (white) and Day 72 (gray) pooled from 2 independent experiments. Box-and-whiskers plots display 25% and 75% percentiles where lines represent maximum, mean, and minimum from top to bottom (n = 6-14). Student’s paired, two-tailed t-tests were used for statistical analysis between time points. (C) Overall survival of mice treated with PBS (dashed), polyclonal (black), Vδ1 (red), Vδ2 (blue), or Vδ1negVδ2neg (green) γδT cells. Log-rank (Mantel-Cox) test was used to calculate p values. *p<0.05, **p<0.01, and ***p≤0.001
See this image and copyright information in PMC

Comment in

"VSports app下载" References

    1. Bonneville M, O’Brien RL, Born WK. Gammadelta T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol. 2010;10:467–78. - PubMed
    1. Vantourout P, Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology. Nat Rev Immunol. 2013;13:88–100. - PMC - PubMed
    1. Lefranc MP. Nomenclature of the human T cell receptor genes. Curr Protoc Immunol. 2001 Appendix 1:Appendix 1O. - "V体育平台登录" PubMed
    1. Turchinovich G, Pennington DJ. T cell receptor signalling in gammadelta cell development: strength isn’t everything. Trends Immunol. 2011;32:567–73. - PubMed
    1. Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology. Cancer Res. 2007;67:5–8. - VSports app下载 - PubMed

Publication types

MeSH terms

Substances