Skip to main page content
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. Before sharing sensitive information, make sure you’re on a federal government site.

Https

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

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jan;60(1):111-22.
doi: 10.1007/s00262-010-0924-z. Epub 2010 Oct 20.

Receptor desensitization and blockade of the suppressive effects of prostaglandin E(2) and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Yunyun Su  1 , Edwin K Jackson, Elieser Gorelik
Affiliations

Receptor desensitization and blockade of the suppressive effects of prostaglandin E(2) and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Yunyun Su et al. Cancer Immunol Immunother. 2011 Jan.

Abstract

Previous studies document that PGE(2) and adenosine suppress production of inflammatory cytokines. The present study demonstrates for the first time that (1) PGE(2) and 2-chloroadenosine (CADO; a stable analog of adenosine) directly inhibit the cytolytic function of human tumor-infiltrating lymphocytes (TILs); (2) the combination PGE(2) and CADO have additive suppressive effects; and (3) the cooperative immunosuppressive actions of PGE(2) and CADO are mediated via EP2 receptors (EP2Rs) and A(2A) receptors (A(2A)Rs) and are due to amplification of cAMP production, activation of protein kinase A (PKA) and T cell receptor (TCR) inhibitor Csk leading to inhibition of Lck, ZAP-70 and Akt phosphorylation. (4) During ex vivo expansion, TILs undergo three stages of differentiation converting from TILs with high cytotoxic activity and relative resistance to combined EP2R/A(2A)R suppression (stage I) to TILs retaining high cytotoxicity and gaining sensitivity to combined suppression (stage II) and then to TILS that are less cytotoxic and very sensitive to combined suppression (stage III). (5) Finally, we find that pretreatment of TILs with non-inhibitory concentrations of EP2R agonists (such as PGE(2) or butaprost) or A(2A)R agonists (such as CADO or CGS21680) increases their cytotoxic activity and induces resistance to EP2R and A(2A)R inhibitory signaling (cross-resistance) due to homologous and heterologous desensitization and internalization of EP2Rs and A(2A)Rs, thus preventing their inhibitory signaling. We conclude that inducing resistance of TILs to the suppressive effects of PGE(2) and adenosine in the tumor microenvironment could represent a novel strategy for improving the efficacy of adoptive immunotherapy.

PubMed Disclaimer

Conflict of interest statement

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1
Combined inhibitory effects of agonists on the cytotoxic activity of TILs. a TILs #2009 were mixed with CADO (50 μM), PGE2 (50 nM), CGS (50 μM), butaprost (5 μM), TGF-β or IL-10 (300 ng/ml) and then 51Cr-labeled T2 cells pulsed with MART-127–35 peptide were added at E:T ratio 5:1. After 4 h of incubation, the level of released radioactivity was determined and percent of cytotoxicity was calculated. Control group significantly (p < 0.01) differs from other groups, except TGF-β or IL-10 treated groups. b TILs #2009 were mixed with 51Cr-labeled UPMCI-Mel-136 melanoma cells at E:T ratio 5:1 in the absence or presence of CGS (25–100 μM) and/or butaprost (5 μM) in a 16 h 51Cr-release assay. Groups with combined CGS + butaprost treatment significantly (p < 0.01) differ from other groups. c Real-time PCR analysis of PGE2 receptors expression in TILs #2009. d Real-time PCR analysis expression of A2A, A2B and EP2, EP4 receptors in TILs #2009
Fig. 2
Fig. 2
Stage-dependent cytotoxic activity of TILs and their sensitivity to the suppressive effects of CGS and butaprost. The cytotoxic activities of four TIL lines (#2009, #2133, #2044, and #3072) were tested against T2 cells pulsed with MART-127–35 peptide in the presence of CGS (50 μM) and/or butaprost (5 μM) at 2–4 weeks intervals during TILs culture with IL-2. *Significantly (p < 0.01) different from control group. **Significantly (p < 0.01) different from other groups
Fig. 3
Fig. 3
Analysis of signaling and cAMP production by TILs at stage I. a The cytotoxic activity of TILs #2009 at stage I was tested against T2 cells pulsed with MART-127–35 peptide in the presence of CADO (50 μM), PGE2 (50 nM), PDE4 inhibitor rolipram (rolipr; 3 μM) or cAMP inducer forskolin (forskl; 25–100 μM). *Significantly (p < 0.05) different from control group. b Effect of CGS (100 μM), butaprost (Buta; 5 μM) and/or rolipram (3 μM) on the cytotoxic activity of TILs #2009 against T2 cells pulsed with MART-127–35 peptide. *Significantly (p < 0.05) different from control group. c The cytotoxic activity of TILs #2009 at stage II was tested against T2 cells pulsed with MART-127–35 peptide in the presence of forskolin (25–100 μM) on the cytotoxic activity of stage II TILs #2009 at E:T ratio 5:1. Inhibitory effect of forskolin was significant (p < 0.01). d, e Analysis of cAMP production. TILs #2009 at stage I were incubated for 10 min with CADO (50 μM), PGE2 (50 nM) and/or, rolipram (3 μM) (d) or with CGS (100 μM), butaprost (5 μM) and/or rolipram (3 μM) (e), and the levels of cAMP in cell extracts were analyzed using mass spectrometry. Combined treatment significantly (p < 0.01) differs from a single agent treatment
Fig. 4
Fig. 4
Effect of CADO and PGE2 on cAMP production (a) and CREB (b) and Csk (c) phosphorylation. a TILs #2009 at stage II were incubated with CADO (50 μM) and/or PGE2 (0.5 μM) or CGS (50 μM) and/or butaprost (5 μM) for 10 min. The levels of cAMP in cellular extracts were analyzed using mass spectrometry. b, c TILs #2009 at stage II were stimulated with anti-CD3 in the presence of CADO (50 μM) and/or PGE2 (0.5 μM) for 30 min. TIL protein extracts (50 μg) were resolved using 10% SDS-PAGE and transferred to PVDF membranes. The level of CREB (b) and Csk (c) phosphorylation was analyzed using specific antibodies. The optic density (OD) ratio of the bands was analyzed using ImageQuanT data analysis software (Molecular Dynamics)
Fig. 5
Fig. 5
Effects of CADO and PGE2 on Lck, ZAP-70 and Akt phosphorylation. a TILs #2009 at stage II were stimulated with anti-CD3 in the presence of CADO (50 μM) and/or PGE2 (0.5 μM) for 30 min. Western blot analysis of Lck phosphorylation was performed using anti-phospho Tyr505 or Tyr394 antibodies. b To analyze Zap-70 phosphorylation, stage II TILs #2009 were stimulated with anti-CD3 Ab. Some TILs were stimulated in the presence of CADO (C 50 μM) and PGE2 (P 0.5 μM) (group αCD3, C + P). Control group: unstimulated TILs. After 30 min of stimulation, all cells were fixed, permeabilized and stained with rabbit anti-phospho-Zap-70 (Tyr493) and then with the secondary anti-rabbit IgG-PE and cell fluorescent intensity was analyzed by flow cytometry. c TILs #2009 were stimulated with anti-CD3 in the presence of CADO (50 μM) and/or PGE2 (0.5 μM) for 30 min. Western blot analysis of Akt phosphorylation was performed using anti-phosphorylated-Akt (Ser473) or anti-total Akt antibody. In non-stimulated TILs, Akt was not phosphorylated (not shown)
See this image and copyright information in PMC

References

    1. Rosenberg SA, Dudley ME. Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol. 2009;21:233–240. doi: 10.1016/j.coi.2009.03.002. - DOI - PMC - PubMed
    1. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med. 2004;10:909–915. doi: 10.1038/nm1100. - DOI - PMC - PubMed
    1. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, Rosenberg SA. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009;114:1537–1544. doi: 10.1182/blood-2008-12-195792. - DOI - PMC - PubMed
    1. Gabrilovich DI. Molecular mechanisms and therapeutic reversal of immune suppression in cancer. Curr Cancer Drug Targets. 2007;7:1. doi: 10.2174/156800907780006850. - DOI - PubMed
    1. Gajewski TF, Meng Y, Blank C, Brown I, Kacha A, Kline J, Harlin H. Immune resistance orchestrated by the tumor microenvironment. Immunol Rev. 2006;213:131–145. doi: 10.1111/j.1600-065X.2006.00442.x. - DOI - PubMed

Publication types

MeSH terms