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A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.

Khandelwal N, Breinig M, Speck T, Michels T, Kreutzer C, Sorrentino A, Sharma AK, Umansky L, Conrad H, Poschke I, Offringa R, König R, Bernhard H, Machlenkin A, Boutros M, Beckhove P - EMBO Mol Med (2015)

Bottom Line: Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells.These ligands represent potent targets for therapeutic inhibition.So far, only few immune-suppressive ligands have been identified.

View Article: PubMed Central - PubMed

Affiliation: Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany n.khandelwal@dkfz.de p.beckhove@dkfz.de.

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Tumor-specific CCR9 impedes Th1-type immune responseA, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.Data information: In all the cases, experiments were performed in triplicate with at least two independent repeats. Mean ± SEM are shown herein, unless stated otherwise, with statistical significance assessed using unpaired, two-tailed Student's t-test. Source data are available online for this figure.
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fig05: Tumor-specific CCR9 impedes Th1-type immune responseA, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.Data information: In all the cases, experiments were performed in triplicate with at least two independent repeats. Mean ± SEM are shown herein, unless stated otherwise, with statistical significance assessed using unpaired, two-tailed Student's t-test. Source data are available online for this figure.

Mentions: We finally explored the influence of CCR9 expression on CTL functions. CCR9 knockdown in MCF7 cells significantly increased the secretion of IFN-γ and granzyme B by survivin-specific CTL in response to MCF7 cells (Fig5A and B), supporting the increased cytotoxicity observed in the kill assays. To assess whether this correlated with increased TCR activation and signaling, we performed TCR phospho-plex analysis in survivin-specific CTLs after contact with CCR9hi or CCR9lo MCF7 cells. With the exception of some degree of reduced Lck phosphorylation (which was detectable only 5 min after exposure to CCR9lo tumor cells), we did not observe any CCR9-dependent changes in TCR signaling (Supplementary Fig S2A and B). Nevertheless, TCR engagement was found to be necessary for CCR9-mediated immunosuppression as polyclonal T cells failed to secrete higher levels of IFN-γ in response to CCR9lo MCF7 cells in the absence of anti-EpCAM x CD3 bi-specific antibody (Supplementary Fig S3). One alternative route of T cell activation is the STAT (signal transducer and activator of transcription) family of transcription factors which regulate cytokine expression in T cells (Yu et al, 2009). CCR9 expressed on MCF7 cells significantly inhibited the secretion of the T-helper-1 (Th1) cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and (to a minor extent) of IFN-γ as well as IL-17, while the secretion of IL-10 was slightly but consistently increased (Fig5C). Accordingly, we observed a significant increase in STAT1 and STAT2 signaling in survivin-specific T cells upon coculture with CCR9lo MCF7 cells, suggesting that anti-tumor type-1 immune response is impeded by tumor-specific CCR9 (Fig5D and E).


A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.

Khandelwal N, Breinig M, Speck T, Michels T, Kreutzer C, Sorrentino A, Sharma AK, Umansky L, Conrad H, Poschke I, Offringa R, König R, Bernhard H, Machlenkin A, Boutros M, Beckhove P - EMBO Mol Med (2015)

Tumor-specific CCR9 impedes Th1-type immune responseA, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.Data information: In all the cases, experiments were performed in triplicate with at least two independent repeats. Mean ± SEM are shown herein, unless stated otherwise, with statistical significance assessed using unpaired, two-tailed Student's t-test. Source data are available online for this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403046&req=5

fig05: Tumor-specific CCR9 impedes Th1-type immune responseA, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.Data information: In all the cases, experiments were performed in triplicate with at least two independent repeats. Mean ± SEM are shown herein, unless stated otherwise, with statistical significance assessed using unpaired, two-tailed Student's t-test. Source data are available online for this figure.
Mentions: We finally explored the influence of CCR9 expression on CTL functions. CCR9 knockdown in MCF7 cells significantly increased the secretion of IFN-γ and granzyme B by survivin-specific CTL in response to MCF7 cells (Fig5A and B), supporting the increased cytotoxicity observed in the kill assays. To assess whether this correlated with increased TCR activation and signaling, we performed TCR phospho-plex analysis in survivin-specific CTLs after contact with CCR9hi or CCR9lo MCF7 cells. With the exception of some degree of reduced Lck phosphorylation (which was detectable only 5 min after exposure to CCR9lo tumor cells), we did not observe any CCR9-dependent changes in TCR signaling (Supplementary Fig S2A and B). Nevertheless, TCR engagement was found to be necessary for CCR9-mediated immunosuppression as polyclonal T cells failed to secrete higher levels of IFN-γ in response to CCR9lo MCF7 cells in the absence of anti-EpCAM x CD3 bi-specific antibody (Supplementary Fig S3). One alternative route of T cell activation is the STAT (signal transducer and activator of transcription) family of transcription factors which regulate cytokine expression in T cells (Yu et al, 2009). CCR9 expressed on MCF7 cells significantly inhibited the secretion of the T-helper-1 (Th1) cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and (to a minor extent) of IFN-γ as well as IL-17, while the secretion of IL-10 was slightly but consistently increased (Fig5C). Accordingly, we observed a significant increase in STAT1 and STAT2 signaling in survivin-specific T cells upon coculture with CCR9lo MCF7 cells, suggesting that anti-tumor type-1 immune response is impeded by tumor-specific CCR9 (Fig5D and E).

Bottom Line: Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells.These ligands represent potent targets for therapeutic inhibition.So far, only few immune-suppressive ligands have been identified.

View Article: PubMed Central - PubMed

Affiliation: Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany n.khandelwal@dkfz.de p.beckhove@dkfz.de.

Show MeSH
Related in: MedlinePlus