The goal of this study was to investigate the effects of adenosine and its stable analogue 2-chloroadenosine (CADO) on the cytotoxic activity and cytokine production by human antimelanoma specific CD8 + and CD4 + T-helper type 1 (Th1) clones. The cytotoxic activity of CD8 + T cells was inhibited by adenosine and CADO. Using Lab MAP multiplex technology, we found that adenosine inhibits production of various cytokines and chemokines by CD8 + and CD4 + T cells. Studies with CGS21680, a specific agonist of adenosine A 2A receptor (AdoRA 2A ), and ZM241385, an AdoRA 2 -selective antagonist, indicate that the inhibitory effects of adenosine are mediated via cyclic AMP (cAMP)-elevating AdoRA 2A , leading to protein kinase A (PKA) activation. Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKAI and PKAII, we found that activation of PKAI, but not of PKAII, mimicked the inhibitory effects of adenosine on T-cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKA inhibitor peptide 14-22) failed to abrogate the inhibitory effects of CADO. In contrast, Rp-8-Br-cAMPS that antagonizes binding of cAMP to the regulatory I subunit and PKA activation was efficient in blocking the inhibitory effect of adenosine on the functional activity of T cells. Our findings on the ability of adenosine to inhibit the effector function of antimelanoma specific T cells suggest that intratumor-produced adenosine could impair the function of tumor-infiltrating T lymphocytes. Thus, blocking the inhibitory activity of tumor-produced adenosine might represent a new strategy for improvement of cancer immunotherapy. [Cancer Res 2007;67(12):5949-56]

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87–111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87–111 stimulates Th1-type and Th-2/Th0-type CD4+ T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4+ T cells and CD4+ T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-γ and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4+ T cells recognizing peptide NY-ESO-1 87–111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87–111 in the large number of cancer patients with NY-ESO-1-expressing tumors.

No abstract

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β2-adrenergic receptor (β2AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β2AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β2AR; however, IR reduced β2AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β2AR and IR, we employed quantitative BRET2 saturation and competition assays. Saturation assay data suggested constitutive β2AR and IR homo- and heteromerization. Calculated acceptor/donor (AD50) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET2 data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β2AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of β2AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β2AR:IR oligomer formation, likely comprising heteromers of homodimers.

EphA2, a member of the receptor tyrosine kinase family, is commonly expressed by a broad range of cancer types, where its level of (over