CoChat
Sign Up
Cited by 164T-cell and B-cell ImmunologyImmunotherapy and Immune ResponsesCancer Immunotherapy and Biomarkers

The DOI matches its Crossref record; 56 of the 56 references it cites were found in a registry or an index.

TLR8 signaling enhances tumor immunity by preventing tumor‐induced T‐cell senescence

Open access
Ye, Jian, Ma, Chunling, Hsueh, Eddy C, Dou, Jie, Mo, Wei, Liu, Shuai, Han, Bing, Huang, Yi, Zhang, Yanping, Varvares, Mark A, Hoft, Daniel F, Peng, Guangyong·2014·EMBO Molecular Medicine
DOI 10.15252/emmm.201403918PMC PMC4287933
Open this paper in CoChat — free account

Abstract

Accumulating evidence suggests the immunosuppressive microenvironments created by malignant tumors represent a major obstacle for effective anti‐tumor immunity. A better understanding of the suppressive mechanisms mediated by tumor microenvironments and the development of strategies to reverse the immune suppression are major challenges for the success of tumor immunotherapy. Here, we report that human tumor cells can induce senescence in naïve/effector T cells, exhibiting potent suppressive function in vitro and in vivo . We further show that tumor‐derived endogenous cyclic adenosine monophosphate (cAMP) is responsible for the induction of T‐cell senescence. Importantly, activation of TLR8 signaling in tumor cells can block the induction and reverse the suppression of senescent naïve and tumor‐specific T cells in vitro and in vivo , resulting in enhanced anti‐tumor immunity. These studies identify a novel mechanism of human tumor‐mediated immune suppression and provide a new strategy to reverse tumor immunosuppressive effects for tumor immunotherapy.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.15252/emmm.201403918

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Tumor cells induce T-cell senescence A, B Increased SA-β-Gal-positive T-cell populations (A) and decreased CD28 expression (B) existed in TILs, compared with those in normal tissue-derived lymphocytes. CD3+ T cells were purified from the cultured tissue-infiltrating T cells from freshly digested tumor or normal tissues by microbeads and SA-β-Gal staining or CD28 expression was determined. BNT: normal breast tissue-derived T cells; BTIL, MTIL, and HNTIL: TIL obtained from breast cancer, melanoma, and head and neck cancers, respectively. C Tumor cell treatment significantly increased SA-β-Gal-positive T-cell populations in co-cultured naïve CD4+ T cells. However, naïve CD4+ T cells cultured in medium only or co-cultured with normal breast cells had little SA-β-Gal expression. Anti-CD3-activated naïve CD4+ T cells were co-cultured with different types of tumor cells or normal breast cells at ratio of 1:1 for 1 day. The treated CD4+ T cells were then separated and stained with SA-β-Gal staining reagents after culture for additional 3 days. Cell lines included: breast cancer (MCF7), melanoma (M586 and M628), colon cancer (SW480 and CC2), ovarian cancer (OC155), prostate cancer (DU145 and PC3), squamous cancers (SSC25 and CAL27), and normal breast cells (BN2 and BN5). D Decreased CD28 expression in naïve CD4+ T cells after culture with different types of tumor cell lines but not normal breast cells. Cell treatment and procedure were the same as in (C). CD28 expression in treated naïve CD4+ T cells were analyzed by flow cytometry. E Increased expression of cell cycle regulatory molecules p53, p21, and p16 in naïve T cells treated with tumor cells. Cell treatment and procedure were the same as in (C). Co-cultured naïve CD4+ T cells were purified and cell lysates were prepared for Western blot analyses. F Suppressive function of tumor-induced senescent T cells. Senescent CD4+ T cells induced by different types of tumor cell lines strongly inhibited the proliferation of responding CD4+ T cells. In contrast, naïve T cells treated or untreated with normal breast cells did not affect the proliferation of responding CD4+ T cells (left panel). Furthermore, senescent CD4+ T cells induced by different types of tumor cell lines did not proliferate themselves (right panel). Cell treatment and procedure were the same as in (C). Treated CD4+ T cells were purified, and the suppressive activity on responding CD4+ T-cell proliferation was evaluated using [3H]-thymidine incorporation assays. Data information: Data are representative of average of three independent experiments ± SD.
Figure 1 Tumor cells induce T-cell senescence A, B Increased SA-β-Gal-positive T-cell populations (A) and decreased CD28 expression (B) existed in TILs, compared with those in normal tissue-derived lymphocytes. CD3+ T cells were purified from the cultured tissue-infiltrating T cells from freshly digested tumor or normal tissues by microbeads and SA-β-Gal staining or CD28 expression was determined. BNT: normal breast tissue-derived T cells; BTIL, MTIL, and HNTIL: TIL obtained from breast cancer, melanoma, and head and neck cancers, respectively. C Tumor cell treatment significantly increased SA-β-Gal-positive T-cell populations in co-cultured naïve CD4+ T cells. However, naïve CD4+ T cells cultured in medium only or co-cultured with normal breast cells had little SA-β-Gal expression. Anti-CD3-activated naïve CD4+ T cells were co-cultured with different types of tumor cells or normal breast cells at ratio of 1:1 for 1 day. The treated CD4+ T cells were then separated and stained with SA-β-Gal staining reagents after culture for additional 3 days. Cell lines included: breast cancer (MCF7), melanoma (M586 and M628), colon cancer (SW480 and CC2), ovarian cancer (OC155), prostate cancer (DU145 and PC3), squamous cancers (SSC25 and CAL27), and normal breast cells (BN2 and BN5). D Decreased CD28 expression in naïve CD4+ T cells after culture with different types of tumor cell lines but not normal breast cells. Cell treatment and procedure were the same as in (C). CD28 expression in treated naïve CD4+ T cells were analyzed by flow cytometry. E Increased expression of cell cycle regulatory molecules p53, p21, and p16 in naïve T cells treated with tumor cells. Cell treatment and procedure were the same as in (C). Co-cultured naïve CD4+ T cells were purified and cell lysates were prepared for Western blot analyses. F Suppressive function of tumor-induced senescent T cells. Senescent CD4+ T cells induced by different types of tumor cell lines strongly inhibited the proliferation of responding CD4+ T cells. In contrast, naïve T cells treated or untreated with normal breast cells did not affect the proliferation of responding CD4+ T cells (left panel). Furthermore, senescent CD4+ T cells induced by different types of tumor cell lines did not proliferate themselves (right panel). Cell treatment and procedure were the same as in (C). Treated CD4+ T cells were purified, and the suppressive activity on responding CD4+ T-cell proliferation was evaluated using [3H]-thymidine incorporation assays. Data information: Data are representative of average of three independent experiments ± SD.CC BY · PubMed Central
Figure 2 Tumor cell-induced T-cell senescence involves the DNA-damage response in senescent T cells Tumor cells induced phosphorylated activation of ATM in naïve CD4+ T cells. Furthermore, treatment with an ATM specific inhibitor KU55933 markedly suppressed the ATM phosphorylation in tumor-treated T cells. Anti-CD3 activated CD4+ T cells were pretreated with or without KU55933 (20 μM) for 1 day and then co-cultured with different types of tumor cells for another 1 day. The co-cultured CD4+ T cells were separated and the p-ATM expression determined after culture for 3 additional days using FACS analyses.Pretreatment of T cells with KU55933 significantly prevented the induction of T-cell senescence induced by different types of tumor cells. Cell treatment procedure was the same as in (A). SA-β-Gal expression in CD4+ T cells was determined with SA-β-Gal staining. Data shown are mean ± SD from three independent experiments, and paired t-test was performed. *P < 0.05, compared with the medium only group.
Figure 2 Tumor cell-induced T-cell senescence involves the DNA-damage response in senescent T cells Tumor cells induced phosphorylated activation of ATM in naïve CD4+ T cells. Furthermore, treatment with an ATM specific inhibitor KU55933 markedly suppressed the ATM phosphorylation in tumor-treated T cells. Anti-CD3 activated CD4+ T cells were pretreated with or without KU55933 (20 μM) for 1 day and then co-cultured with different types of tumor cells for another 1 day. The co-cultured CD4+ T cells were separated and the p-ATM expression determined after culture for 3 additional days using FACS analyses.Pretreatment of T cells with KU55933 significantly prevented the induction of T-cell senescence induced by different types of tumor cells. Cell treatment procedure was the same as in (A). SA-β-Gal expression in CD4+ T cells was determined with SA-β-Gal staining. Data shown are mean ± SD from three independent experiments, and paired t-test was performed. *P < 0.05, compared with the medium only group.CC BY · PubMed Central
Figure 3 Tumor-induced T-cell senescence is dependent on cell–cell contact, but independent of known suppressive cytokines and inhibitory molecules Significant decrease of the SA-β-Gal-positive T-cell populations in naïve CD4+ T cells induced by tumor cells after separation by a Transwell system. Anti-CD3-activated naïve CD4+ T cells were either co-cultured with tumor cells as described in Fig 1 or were separated with tumor cells by a Transwell system in 24-well plates with pore size 0.4 μm insert chambers. SA-β-Gal expression was measured in the treated CD4+ T cells. Data shown are mean ± SD from three independent experiments, and paired t-test was used. **P < 0.01, compared with the naïve T cells in the co-culture system.Separation of naïve CD4+ T cells and tumor cells by a Transwell system alleviated the loss of CD28 expression in tumor-treated T cells. Cell treatment and procedure were the same as in (A). CD28 expression in treated naïve CD4+ T cells were analyzed by flow cytometry.Induction of T-cell senescence mediated by tumor cells cannot be prevented through the blockage of IL-10, TGF-β, TCRαβ, PDL-1 and MHC-class II and IDO pathways. Tumor cells and naïve CD4+ T cells were pretreated with various indicated neutralizing antibodies and the IDO inhibitor 1-MT and then co-cultured in the presence or absence of these neutralizing antibodies and 1-MT. The co-cultured CD4+ T cells were then separated and analyzed for SA-β-Gal expression.
Figure 3 Tumor-induced T-cell senescence is dependent on cell–cell contact, but independent of known suppressive cytokines and inhibitory molecules Significant decrease of the SA-β-Gal-positive T-cell populations in naïve CD4+ T cells induced by tumor cells after separation by a Transwell system. Anti-CD3-activated naïve CD4+ T cells were either co-cultured with tumor cells as described in Fig 1 or were separated with tumor cells by a Transwell system in 24-well plates with pore size 0.4 μm insert chambers. SA-β-Gal expression was measured in the treated CD4+ T cells. Data shown are mean ± SD from three independent experiments, and paired t-test was used. **P < 0.01, compared with the naïve T cells in the co-culture system.Separation of naïve CD4+ T cells and tumor cells by a Transwell system alleviated the loss of CD28 expression in tumor-treated T cells. Cell treatment and procedure were the same as in (A). CD28 expression in treated naïve CD4+ T cells were analyzed by flow cytometry.Induction of T-cell senescence mediated by tumor cells cannot be prevented through the blockage of IL-10, TGF-β, TCRαβ, PDL-1 and MHC-class II and IDO pathways. Tumor cells and naïve CD4+ T cells were pretreated with various indicated neutralizing antibodies and the IDO inhibitor 1-MT and then co-cultured in the presence or absence of these neutralizing antibodies and 1-MT. The co-cultured CD4+ T cells were then separated and analyzed for SA-β-Gal expression.CC BY · PubMed Central
Figure 4 Tumor-derived endogenous cAMP is a key mediator inducing T-cell senescence cAMP levels in M628, PC3, and MCF7 tumor cells were significantly higher than those in normal breast cells. Concentrations of cAMP in cell lysates were determined by ELISA.cAMP levels in CD4+ T cells were significantly increased after co-culture with M628, MCF7, or PC-3 tumor cells, whereas its levels in CD4+ T cells were dramatically decreased if the tumors and T cells were co-cultured in the presence of the gap junction inhibitory peptide GAP27. Tumor cells were co-cultured with anti-CD3-activated CD4+ T cells in the presence or absence of GAP27 (300 μM) for 1 day. The treated T cells were separated and the cAMP levels determined after culture for additional 3 days.Blocking gap junction formation with GAP27 significantly prevented tumor-induced CD4+ T-cell senescence. Cell treatment and procedure were the same as in (B). SA-β-Gal expression was measured in the treated CD4+ T cells.PC3 and MCF7 tumor cells induced phosphorylation of LCK, CREB, and PKA in senescent CD4+ T cells. Anti-CD3-activated CD4+ T cells were co-cultured with MCF7 or PC3 for 0, 1, 3, and 5 days. Co-cultured naïve CD4+ T cells were purified and then lysates prepared for Western blot analyses.Inhibition of cAMP signaling by specific pharmacological inhibitors significantly reversed T-cell senescence induced by tumor cells. Anti-CD3 preactivated naïve CD4+ T cells were co-cultured with tumor cells in the presence or absence of inhibitors 7-ddA (320 μM), or H89 (20 μM) or the combination for 1 day. Co-cultured CD4+ T cells were separated, and SA-β-Gal expression was determined after culture for an additional 3 days.Pretreatment of tumor cells with the 7-ddA and H89 markedly decreased cAMP levels in co-cultured CD4+ T cells. MCF7 or PC-3 cells were pretreated with or without 7-ddA (320 μM) and H89 (20 μM) for 1 day, washed, and then co-cultured with anti-CD3 activated CD4+ T cells for 1 day. The co-cultured CD4+ T cells were purified and cAMP levels determined. Data information: Data shown are mean ± SD from three independent experiments and paired t-test was performed between groups. *P < 0.05 and **P < 0.01, compared with the medium only group (B, C, and F), or with the group not treated with inhibitors (E).
Figure 4 Tumor-derived endogenous cAMP is a key mediator inducing T-cell senescence cAMP levels in M628, PC3, and MCF7 tumor cells were significantly higher than those in normal breast cells. Concentrations of cAMP in cell lysates were determined by ELISA.cAMP levels in CD4+ T cells were significantly increased after co-culture with M628, MCF7, or PC-3 tumor cells, whereas its levels in CD4+ T cells were dramatically decreased if the tumors and T cells were co-cultured in the presence of the gap junction inhibitory peptide GAP27. Tumor cells were co-cultured with anti-CD3-activated CD4+ T cells in the presence or absence of GAP27 (300 μM) for 1 day. The treated T cells were separated and the cAMP levels determined after culture for additional 3 days.Blocking gap junction formation with GAP27 significantly prevented tumor-induced CD4+ T-cell senescence. Cell treatment and procedure were the same as in (B). SA-β-Gal expression was measured in the treated CD4+ T cells.PC3 and MCF7 tumor cells induced phosphorylation of LCK, CREB, and PKA in senescent CD4+ T cells. Anti-CD3-activated CD4+ T cells were co-cultured with MCF7 or PC3 for 0, 1, 3, and 5 days. Co-cultured naïve CD4+ T cells were purified and then lysates prepared for Western blot analyses.Inhibition of cAMP signaling by specific pharmacological inhibitors significantly reversed T-cell senescence induced by tumor cells. Anti-CD3 preactivated naïve CD4+ T cells were co-cultured with tumor cells in the presence or absence of inhibitors 7-ddA (320 μM), or H89 (20 μM) or the combination for 1 day. Co-cultured CD4+ T cells were separated, and SA-β-Gal expression was determined after culture for an additional 3 days.Pretreatment of tumor cells with the 7-ddA and H89 markedly decreased cAMP levels in co-cultured CD4+ T cells. MCF7 or PC-3 cells were pretreated with or without 7-ddA (320 μM) and H89 (20 μM) for 1 day, washed, and then co-cultured with anti-CD3 activated CD4+ T cells for 1 day. The co-cultured CD4+ T cells were purified and cAMP levels determined. Data information: Data shown are mean ± SD from three independent experiments and paired t-test was performed between groups. *P < 0.05 and **P < 0.01, compared with the medium only group (B, C, and F), or with the group not treated with inhibitors (E).CC BY · PubMed Central
Figure 5 TLR8 signaling prevents the induction of CD4+ T-cell senescence mediated by tumor cells TLR8 ligands Poly-G3 and ssRNA40 markedly reversed the ability of tumor cells to induce naïve CD4+ T-cell senescence. MCF7, PC3, and M628 cells were co-cultured with anti-CD3 activated CD4+ T cells in the presence or absence of the indicated TLR ligands for 1 day. The treated CD4+ T cells were then separated and SA-β-Gal expression determined after culture for additional 3 days. Poly-T3 (3 μg/ml) served as a control.TLR ligands did not induce senescence in CD4+ T cells. Preactivated CD4+ T cells freshly purified from two healthy donors were cultured in the presence of the indicated TLR ligands for 3 days and SA-β-Gal-positive CD4+ T cells determined.Knockdown of TLR8, MyD88, or IRAK4 in MCF7, M628, or PC3 tumor cells blocked the reversal of tumor-induced CD4+ T-cell senescence mediated by Poly-G3. Tumor cells were transfected with lenti-shRNAs specific for TLR8, MyD88, or IRAK4 molecules. Transduced (GFP+) tumor cells were purified by FACS sorting and co-cultured with anti-CD3 activated CD4+ T cells in the presence or absence of Poly-G3 for 1 day. The treated CD4+ T cells were then separated, and the SA-β-Gal expression was determined after culture for additional 3 days.Pretreatment of PC3 or M628 tumor cells with the inhibitor U0126, and MCF7 cells with both inhibitors U0126 and SB203580, significantly blocked the Poly-G3-mediated reversal of tumor-induced T-cell senescence. Tumor cells were pretreated for 3 days with MAPK inhibitors U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM), or the NF-κB inhibitor APDC (10 μM), and Poly-G3 was added into co-cultures on day 3. The pretreated tumor cells were co-cultured with anti-CD3-activated CD4+ T cells at ratio of 1:1 for 1 day. The treated T cells were separated and cultured for additional 3 days, and then SA-β-Gal expression assessed. Data information: Data are average (A) or mean (C, D) of three independent experiments ± SD, and paired t-test was used between groups. *P < 0.05 and **P < 0.01, compared with the group not treated with TLR ligand (A), with the group transfected with control shRNA (C), or with the group not treated with inhibitor (D).
Figure 5 TLR8 signaling prevents the induction of CD4+ T-cell senescence mediated by tumor cells TLR8 ligands Poly-G3 and ssRNA40 markedly reversed the ability of tumor cells to induce naïve CD4+ T-cell senescence. MCF7, PC3, and M628 cells were co-cultured with anti-CD3 activated CD4+ T cells in the presence or absence of the indicated TLR ligands for 1 day. The treated CD4+ T cells were then separated and SA-β-Gal expression determined after culture for additional 3 days. Poly-T3 (3 μg/ml) served as a control.TLR ligands did not induce senescence in CD4+ T cells. Preactivated CD4+ T cells freshly purified from two healthy donors were cultured in the presence of the indicated TLR ligands for 3 days and SA-β-Gal-positive CD4+ T cells determined.Knockdown of TLR8, MyD88, or IRAK4 in MCF7, M628, or PC3 tumor cells blocked the reversal of tumor-induced CD4+ T-cell senescence mediated by Poly-G3. Tumor cells were transfected with lenti-shRNAs specific for TLR8, MyD88, or IRAK4 molecules. Transduced (GFP+) tumor cells were purified by FACS sorting and co-cultured with anti-CD3 activated CD4+ T cells in the presence or absence of Poly-G3 for 1 day. The treated CD4+ T cells were then separated, and the SA-β-Gal expression was determined after culture for additional 3 days.Pretreatment of PC3 or M628 tumor cells with the inhibitor U0126, and MCF7 cells with both inhibitors U0126 and SB203580, significantly blocked the Poly-G3-mediated reversal of tumor-induced T-cell senescence. Tumor cells were pretreated for 3 days with MAPK inhibitors U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM), or the NF-κB inhibitor APDC (10 μM), and Poly-G3 was added into co-cultures on day 3. The pretreated tumor cells were co-cultured with anti-CD3-activated CD4+ T cells at ratio of 1:1 for 1 day. The treated T cells were separated and cultured for additional 3 days, and then SA-β-Gal expression assessed. Data information: Data are average (A) or mean (C, D) of three independent experiments ± SD, and paired t-test was used between groups. *P < 0.05 and **P < 0.01, compared with the group not treated with TLR ligand (A), with the group transfected with control shRNA (C), or with the group not treated with inhibitor (D).CC BY · PubMed Central
Figure 6 TLR8 signaling reverses tumor-induced T-cell senescence via down-regulation of cAMP in tumor cells A Pretreatment of tumor cells with Poly-G3 down-regulated the expression of p53, p21, and p16 in tumor-induced senescent CD4+ T cells. CD4+ T cells were co-cultured with Poly-G3-pretreated or untreated tumor cells (MCF7, PC3, or M628) for 1 day. The cultured CD4+ T cells were separated, and p53, p21, and p16 expression was determined using Western blot analysis after culture for 3 additional days. B Pretreatment of MCF7 cells with Poly-G3, but not with Poly-T3 (control), significantly reversed the suppressive activity on the responding T-cell proliferation mediated by tumor-treated naïve CD4+ T cells. Naive CD4+ T cells were co-cultured with BN5 or MCF7 cells pretreated with or without Poly-G3 for 1 day. Treated CD4+ were separated, and the suppressive activities on responding CD4+ T-cell proliferation were evaluated using [3H]-thymidine incorporation assays. C, D Poly-G3 treatment significantly decreased cAMP levels in tumor cells (C) and in tumor-induced senescent CD4+ T cells (D). MCF7 or PC-3 cells were co-cultured with Anti-CD3-activated CD4+ T cells for 1 day in the presence or absence of Poly-G3 (3 μg/ml). cAMP levels in tumor cells and in co-cultured CD4+ T cells were detected. E Knockdown of ERK1/2 and p38α in MCF7, and ERK1/2 in PC3 tumor cells significantly blocked the Poly-G3-mediated down-regulation of cAMP levels in tumor cells. Tumor cells were transfected with lenti-shRNAs specific for ERK1, ERK2 or p38α molecules or control shRNA. Transduced (GFP+) tumor cells were purified by FACS sorting and cultured for 1 day in the presence of Poly-G3 (3 μg/ml). cAMP levels in tumor cells were detected. Data information: Data shown are mean of three independent experiments ± SD, and paired t-test was performed between groups. **P < 0.01, compared with the group transfected with control shRNA (E).
Figure 6 TLR8 signaling reverses tumor-induced T-cell senescence via down-regulation of cAMP in tumor cells A Pretreatment of tumor cells with Poly-G3 down-regulated the expression of p53, p21, and p16 in tumor-induced senescent CD4+ T cells. CD4+ T cells were co-cultured with Poly-G3-pretreated or untreated tumor cells (MCF7, PC3, or M628) for 1 day. The cultured CD4+ T cells were separated, and p53, p21, and p16 expression was determined using Western blot analysis after culture for 3 additional days. B Pretreatment of MCF7 cells with Poly-G3, but not with Poly-T3 (control), significantly reversed the suppressive activity on the responding T-cell proliferation mediated by tumor-treated naïve CD4+ T cells. Naive CD4+ T cells were co-cultured with BN5 or MCF7 cells pretreated with or without Poly-G3 for 1 day. Treated CD4+ were separated, and the suppressive activities on responding CD4+ T-cell proliferation were evaluated using [3H]-thymidine incorporation assays. C, D Poly-G3 treatment significantly decreased cAMP levels in tumor cells (C) and in tumor-induced senescent CD4+ T cells (D). MCF7 or PC-3 cells were co-cultured with Anti-CD3-activated CD4+ T cells for 1 day in the presence or absence of Poly-G3 (3 μg/ml). cAMP levels in tumor cells and in co-cultured CD4+ T cells were detected. E Knockdown of ERK1/2 and p38α in MCF7, and ERK1/2 in PC3 tumor cells significantly blocked the Poly-G3-mediated down-regulation of cAMP levels in tumor cells. Tumor cells were transfected with lenti-shRNAs specific for ERK1, ERK2 or p38α molecules or control shRNA. Transduced (GFP+) tumor cells were purified by FACS sorting and cultured for 1 day in the presence of Poly-G3 (3 μg/ml). cAMP levels in tumor cells were detected. Data information: Data shown are mean of three independent experiments ± SD, and paired t-test was performed between groups. **P < 0.01, compared with the group transfected with control shRNA (E).CC BY · PubMed Central
Figure 7 Human melanoma cells induce naïve CD4+ T-cell senescence in vivo A Increased SA-β-Gal-positive cell populations were markedly induced in preactivated human CD4+ T cells after adoptive transfer into the tumor 586mel-bearing Rag1−/− mice but not in control Rag1−/− mice. Human naïve CD4+ T cells (5 × 106/mouse) were preactivated with anti-CD3 antibody and adoptively transferred into control Rag1−/− mice or human 586mel-bearing Rag1−/− mice. The transferred human CD4+ T cells were isolated and recovered from the blood, LNs and SPs, as well as tumor tissues (tumor-bearing mice) at 12 days post-injection for subsequent SA-β-Gal staining. B cAMP levels were significantly increased in CD4+ T cells after adoptive transfer into the tumor 586mel-bearing Rag1−/− mice but not in control Rag1−/− mice. Cell treatment and adoptive transfer procedure were identical as in (A). C The transferred human CD4+ T cells isolated and recovered from different organs and tumors in tumor 586mel-bearing mice had potent suppressive activity on the proliferation of responding CD4+ T cells using 3H-thymidine incorporation assays. D, E Intratumoral injection of Poly-G3 (50 μg in 100 μl PBS/mouse), but not LPS (10 μg in 100 μl PBS/mouse) or PBS (100 μl/mouse), can significantly block the induction of senescence and reverse the suppressive activity in transferred naïve CD4+ T cells in human 586mel-bearing mice. Cell preparation and injection procedures were the same as in (A). The transferred human CD4+ T cells in different organs were isolated at 12 days post-injection for subsequent SA-β-Gal staining (D) and 3H-thymidine incorporation assays (E). Data information: Results shown are mean ± SD (n = 5 mice per group). **P < 0.01, compared with the respective CD4+ T-cell group recovered from control Rag1−/− mice (A–C); #P < 0.01, compared with the average levels of CD4+ T-cell groups recovered from control Rag1−/− mice (A–C); *P < 0.05 compared with the PBS group (D, E) using unpaired t-test.
Figure 7 Human melanoma cells induce naïve CD4+ T-cell senescence in vivo A Increased SA-β-Gal-positive cell populations were markedly induced in preactivated human CD4+ T cells after adoptive transfer into the tumor 586mel-bearing Rag1−/− mice but not in control Rag1−/− mice. Human naïve CD4+ T cells (5 × 106/mouse) were preactivated with anti-CD3 antibody and adoptively transferred into control Rag1−/− mice or human 586mel-bearing Rag1−/− mice. The transferred human CD4+ T cells were isolated and recovered from the blood, LNs and SPs, as well as tumor tissues (tumor-bearing mice) at 12 days post-injection for subsequent SA-β-Gal staining. B cAMP levels were significantly increased in CD4+ T cells after adoptive transfer into the tumor 586mel-bearing Rag1−/− mice but not in control Rag1−/− mice. Cell treatment and adoptive transfer procedure were identical as in (A). C The transferred human CD4+ T cells isolated and recovered from different organs and tumors in tumor 586mel-bearing mice had potent suppressive activity on the proliferation of responding CD4+ T cells using 3H-thymidine incorporation assays. D, E Intratumoral injection of Poly-G3 (50 μg in 100 μl PBS/mouse), but not LPS (10 μg in 100 μl PBS/mouse) or PBS (100 μl/mouse), can significantly block the induction of senescence and reverse the suppressive activity in transferred naïve CD4+ T cells in human 586mel-bearing mice. Cell preparation and injection procedures were the same as in (A). The transferred human CD4+ T cells in different organs were isolated at 12 days post-injection for subsequent SA-β-Gal staining (D) and 3H-thymidine incorporation assays (E). Data information: Results shown are mean ± SD (n = 5 mice per group). **P < 0.01, compared with the respective CD4+ T-cell group recovered from control Rag1−/− mice (A–C); #P < 0.01, compared with the average levels of CD4+ T-cell groups recovered from control Rag1−/− mice (A–C); *P < 0.05 compared with the PBS group (D, E) using unpaired t-test.CC BY · PubMed Central
Figure 8 Enhancement of anti-tumor immunity mediated by tumor-specific CD8+ T cells protected against tumor-induced senescence via TLR8 signaling in vivo A SA-β-Gal-positive cell populations were markedly induced in human tumor-specific CD8+ TIL586 cells after adoptive transfer into the tumor 586mel–bearing NOD-scid Il2rgnull (NSG) mice but not into control NSG mice. Human CD8+ TIL586 cells (5 × 106/mouse) were adoptively transferred into control or human 586mel-bearing NSG mice. Blood, LNs and SPs, as well as tumor tissues (tumor-bearing mice) were harvested at 12 days post-injection. The transferred human CD8+ TIL586 were isolated and recovered for subsequent SA-β-Gal staining. B The purified CD8+ TIL586 cells transferred into 586mel-bearing mice became suppressive T cells and had suppressive activity on the proliferation of responding CD4+ T cells. Cell treatment and adoptive transfer procedure were identical as in (A). The transferred human CD8+ TIL586 were isolated and recovered for subsequent 3H-thymidine incorporation assays. C, D Intratumoral injection of Poly-G3 but not LPS can significantly block the induction of senescence and reverse the suppressive activity in transferred CD8+ TIL586 cells in human 586mel-bearing mice. Cell preparation and injection procedures were the same as in (A). PBS (100 μl/mouse), LPS (10 μg in 100 μl PBS/mouse), or Poly-G3 (50 μg in 100 μl PBS/mouse) was injected into tumors on days 1, 4, 7, and 10 after adoptive transfer of CD8+ TIL586 cells into the mice. The transferred human CD8+ TIL586 cells in different organs were isolated at 12 days post-injection for subsequent SA-β-Gal staining (C) and 3H-thymidine incorporation assays (D). E Intratumoral injection of Poly-G3 enhanced anti-tumor immunity in NSG mice. Human 586mel tumor cells were subcutaneously injected into NSG on day 0. Tumor-specific CD8+ TIL586 cells were injected i.v. on day 3 with or without intratumoral injection of Poly-G3. Tumor volumes were measured and presented as mean ± SD (n = 5 mice per group). P-values were determined by the one-way analysis of variance (ANOVA). Similar results were obtained in three repeat experiments. Data information: Results shown are mean ± SD (n = 5 mice per group). **P < 0.01, compared with the respective CD8+ TIL586 group recovered from control NSG mice (A, B); #P < 0.01, compared with the average levels of CD8+ TIL586 group recovered from control NSG mice (A, B); *P < 0.05 and **P < 0.01, compared with the PBS group (C, D) using unpaired t-test.
Figure 8 Enhancement of anti-tumor immunity mediated by tumor-specific CD8+ T cells protected against tumor-induced senescence via TLR8 signaling in vivo A SA-β-Gal-positive cell populations were markedly induced in human tumor-specific CD8+ TIL586 cells after adoptive transfer into the tumor 586mel–bearing NOD-scid Il2rgnull (NSG) mice but not into control NSG mice. Human CD8+ TIL586 cells (5 × 106/mouse) were adoptively transferred into control or human 586mel-bearing NSG mice. Blood, LNs and SPs, as well as tumor tissues (tumor-bearing mice) were harvested at 12 days post-injection. The transferred human CD8+ TIL586 were isolated and recovered for subsequent SA-β-Gal staining. B The purified CD8+ TIL586 cells transferred into 586mel-bearing mice became suppressive T cells and had suppressive activity on the proliferation of responding CD4+ T cells. Cell treatment and adoptive transfer procedure were identical as in (A). The transferred human CD8+ TIL586 were isolated and recovered for subsequent 3H-thymidine incorporation assays. C, D Intratumoral injection of Poly-G3 but not LPS can significantly block the induction of senescence and reverse the suppressive activity in transferred CD8+ TIL586 cells in human 586mel-bearing mice. Cell preparation and injection procedures were the same as in (A). PBS (100 μl/mouse), LPS (10 μg in 100 μl PBS/mouse), or Poly-G3 (50 μg in 100 μl PBS/mouse) was injected into tumors on days 1, 4, 7, and 10 after adoptive transfer of CD8+ TIL586 cells into the mice. The transferred human CD8+ TIL586 cells in different organs were isolated at 12 days post-injection for subsequent SA-β-Gal staining (C) and 3H-thymidine incorporation assays (D). E Intratumoral injection of Poly-G3 enhanced anti-tumor immunity in NSG mice. Human 586mel tumor cells were subcutaneously injected into NSG on day 0. Tumor-specific CD8+ TIL586 cells were injected i.v. on day 3 with or without intratumoral injection of Poly-G3. Tumor volumes were measured and presented as mean ± SD (n = 5 mice per group). P-values were determined by the one-way analysis of variance (ANOVA). Similar results were obtained in three repeat experiments. Data information: Results shown are mean ± SD (n = 5 mice per group). **P < 0.01, compared with the respective CD8+ TIL586 group recovered from control NSG mice (A, B); #P < 0.01, compared with the average levels of CD8+ TIL586 group recovered from control NSG mice (A, B); *P < 0.05 and **P < 0.01, compared with the PBS group (C, D) using unpaired t-test.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 56 references

Cited by

Sorted by citations

See all 164

Related

Metadata

Authors
Ye, J., Ma, C., Hsueh, E. C., Dou, J., Mo, W., Liu, S., Han, B., Huang, Y., Zhang, Y., Varvares, M. A., Hoft, D. F., & Peng, G.
Affiliations
Saint Louis University (US)
Corresponding author
Peng, Guangyong
ORCID
Venue
EMBO Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2014-09-17
Volume
6
Issue
10
Pages
1294-1311
ISSN
  • 1757-4676 (ISSN-L)
  • 1757-4684
DOI
10.15252/emmm.201403918
PMC
PMC4287933
OpenAlex
W2000656984
Cited by
161
References
56
Citations by year
2026: 6 · 2025: 24 · 2024: 24 · 2023: 14 · 2022: 16 · 2021: 27 · 2020: 22 · 2019: 6 · 2018: 13 · 2017: 5 · 2016: 1 · 2015: 3
FWCI
1.37
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Molecular Medicine (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • American Cancer Society (RSG‐10‐160‐01‐LIB)
  • Melanoma Research Alliance
  • National Institutes of Health
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
Keywords
T cell senescence, tumor immunosuppression, tumor microenvironment, cyclic AMP, antitumor immunity, cancer immunotherapy, tumor-specific T cells
MeSH terms
Cyclic AMP, Animals, Cells, Cultured, Flow Cytometry, Humans, Neoplasms, T-Lymphocytes, Blotting, Western, Signal Transduction, CD4-Positive T-Lymphocytes, Immunotherapy, Adoptive, Mice, SCID, Mice, Inbred NOD, Cellular Senescence, Mice, Knockout, Coculture Techniques, Xenograft Model Antitumor Assays, RNA Interference, Cell Line, Tumor, Toll-Like Receptor 8, Interleukin Receptor Common gamma Subunit, Tumor Microenvironment, MCF-7 Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review