Salem Chouaib

PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

Hypoxia selectively up-regulates PD-L1 on myeloid-derived suppressor cells via HIF-1a, thus affecting T cell activation.

The host—tumor immune conflict: from immunosuppression to resistance and destruction

A successful immune response against a tumor is dependent on the cytokine repertoire present at the tumor site. Salem Chouaib and colleagues discuss evidence that, to escape the immune system, tumor cells not only produce immunosuppressive cytokines but also employ strategies involving altered susceptibility to tumor necrosis factor and Fas cytotoxic pathways and, in some circumstances, use of the Fas ligand to neutralize effector cells.

Phosphostim-Activated γδ T Cells Kill Autologous Metastatic Renal Cell Carcinoma

Metastatic renal cell carcinoma, inherently resistant to conventional treatments, is considered immunogenic. Indeed, partial responses are obtained after treatment with cytokines such as IL-2 or IFN-α, suggesting that the immune system may control the tumor growth. In this study, we have investigated the ability of the main subset of peripheral γδ lymphocytes, the Vγ9Vδ2-TCR T lymphocytes, to induce an effective cytotoxic response against autologous primary renal cell carcinoma lines. These γδ T cells were expanded ex vivo using a Vγ9Vδ2 agonist, a synthetic phosphoantigen called Phosphostim. From 11 of 15 patients, the peripheral Vγ9Vδ2 T cells were amplified in vitro by stimulating PBMCs with IL-2 and Phosphostim molecule. These expanded Vγ9Vδ2 T cells express activation markers and exhibit an effector/memory phenotype. They display a selective lytic potential toward autologous primary renal tumor cells and not against renal NC. The lytic activity involves the perforin-granzyme pathway and is mainly TCR and NKG2D receptor dependent. Furthermore, an increased expression of MHC class I-related molecule A or B proteins, known ligands of NKG2D, are detected on primary renal tumor cells. Interestingly, from 2 of the 11 positive cultures in response to Phosphostim, expanded-Vγ9Vδ2 T cells present an expression of killer cell Ig-like receptors, suggesting their prior recruitment in vivo. Unexpectedly, on serial frozen sections from three tumors, we observe a γδ lymphocyte infiltrate that was mainly composed of Vγ9Vδ2 T cells. These results outline that Vγ9Vδ2-TCR effectors may represent a promising approach for the treatment of metastatic renal cell carcinoma.

Quantitative analysis of Th1, Th2 and TGF-β1 cytokine expression in tumor, TIL and PBL of non-small cell lung cancer patients

For understanding the local immune response in human non‐small cell lung cancer (NSCLC), we investigated both Th1 and Th2‐type as well as TGF‐β1 cytokine mRNA expression in 10 fresh tumor biopsies, the corresponding tumor and short term TIL cell lines as well as patient PBMC. A methodology based on a highly sensitive quantitative RT‐PCR was used. We found that IL‐6 mRNA was highly expressed in all tumor biopsy samples analyzed (4 LLC, 3 ADC and 3 SCC). IL‐10 mRNA was expressed in 7 of 10 biopsies whereas IL‐4 mRNA expression was moderate. Analysis of type 1 cytokines revealed a low expression level of IL‐2 mRNA, while IFNγ and GM‐CSF expression was high in the majority of the tumor lesions studied. Quantitatively, high amounts of Th2‐type cytokine mRNA were detected at the tumor site with IL‐6 as the predominant lymphokine. A high mRNA expression level of the immunosuppressive cytokine TGF‐β1 was observed in all NSCLC. To identify the cell types responsible for the production of TGF‐β1, IL‐6, IL‐10 and GM‐CSF at the tumor site, tumor and TIL cell lines were derived from the corresponding biopsies. All the 3 tumor cell lines analysed were found to express high amount of TGF‐β1 but not IL‐10 mRNA, 2 expressing IL‐6 and GM‐CSF. Five short term TIL cell lines established in the presence of IL‐2 expressed high level of IL‐10, IL‐4 and IFNγ but not IL‐2 mRNA. Strikingly, high expression of IL‐10 mRNA was also observed in all 6 patient PBMC analyzed as compared to controls. Together, our results indicate the existence of a local and peripheral Th‐2‐type cytokine pattern in patients bearing NSCLC. Int. J. Cancer 77:7–12, 1998.© 1998 Wiley‐Liss, Inc.

HER-2/neu and hTERT Cryptic Epitopes as Novel Targets for Broad Spectrum Tumor Immunotherapy

Tolerance to tumor-nonmutated self proteins represents a major obstacle for successful cancer immunotherapy. Since this tolerance primarily concerns dominant epitopes, we hypothesized that targeting cryptic epitopes that have a low affinity for HLA could be an efficient strategy to breach the tolerance to tumor Ags. Using the P1Y heteroclitic peptide approach, we identified low affinity cryptic HLA-A*0201-restricted epitopes derived from two widely expressed tumor Ags, HER-2/neu and hTERT. The P1Y variants of four HER-2/neu (neu391, neu402, neu466, neu650)- and two hTERT (hTERT572 and hTERT988)-derived low affinity peptides exhibited strong affinity for HLA-A*0201 and stimulated specific CTL from healthy donor PBMCs. These CTL specifically recognized HER-2/neu- and hTERT-expressing tumor cells of various histological origins. In vivo studies showed that HLA-A*0201 transgenic HHD mice vaccinated with the P1Y variant peptides generated CTL that specifically lysed Ag-expressing tumor cells, thus recognizing the cognate endogenous Ags. These results suggest that heteroclitic variants of low affinity, cryptic epitopes of widely expressed tumor Ags may serve as valid tools for tumor immunotherapy.

Epithelial-to-mesenchymal transition and autophagy induction in breast carcinoma promote escape from T-cell-mediated lysis.

Epithelial-to-mesenchymal transition (EMT) mediates cancer cell invasion, metastasis, and drug resistance, but its impact on immune surveillance has not been explored. In this study, we investigated the functional consequences of this mode of epithelial cell plasticity on targeted cell lysis by cytotoxic T lymphocytes (CTL). Acquisition of the EMT phenotype in various derivatives of MCF-7 human breast cancer cells was associated with dramatic morphologic changes and actin cytoskeleton remodeling, with CD24(-)/CD44(+)/ALDH(+) stem cell populations present exhibiting a higher degree of EMT relative to parental cells. Strikingly, acquisition of this phenotype also associated with an inhibition of CTL-mediated tumor cell lysis. Resistant cells exhibited attenuation in the formation of an immunologic synapse with CTLs along with the induction of autophagy in the target cells. This response was critical for susceptibility to CTL-mediated lysis because siRNA-mediated silencing of beclin1 to inhibit autophagy in target cells restored their susceptibility to CTL-induced lysis. Our results argue that in addition to promoting invasion and metastasis EMT also profoundly alters the susceptibility of cancer cells to T-cell-mediated immune surveillance. Furthermore, they reveal EMT and autophagy as conceptual realms for immunotherapeutic strategies to block immune escape.

IN VITRO PROCESSING OF HUMAN TUMOR NECROSIS FACTOR-ALPHA

Tumor necrosis factor (TNF)-α is initially synthesized as a membrane-bound, cell-associated 26-kDa protein that is further cleaved to yield the soluble 17-kDa form. By using a radiolabeled in vitro translated TNF-α precursor we detected a serine proteinase processing activity present in crude membrane preparations of monocytic cells able to generate a 17-kDa active protein. A similar processing pattern was obtained using purified neutral serine proteinase proteinase-3 (PR-3). Moreover, while a secretory leukocyte proteinase inhibitor (a natural serine anti-proteinase) did not affect the in vitro TNF-α processing, IgG preparations containing high titers of anti-PR-3 autoantibodies completely blocked this activity. The NH2-terminal sequencing of the reaction products obtained with either membrane preparations or PR-3 showed that cleavage occurs in both cases between Val77 and Arg78. These results together with cellular expression and localization of PR-3 suggest a potential role for this enzyme as an accessory TNF-α processing enzyme.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T cell activity and promotes regression

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Antiviral agent Cidofovir restores p53 function and enhances the radiosensitivity in HPV-associated cancers.

High-risk human papillomaviruses (HPVs) have been associated to the development of cervical and some other human cancers. Most of them express E6 and E7 oncoproteins, able to bind to p53 and retinoblastoma (pRb) tumor suppressor proteins respectively and neutralize their function. Restoration of these pathways by blocking E6 and E7 expression would provide a selective therapeutic effect. Here, we show that a clinically approved antiviral agent Cidofovir reduced E6 and E7 expression in cervical carcinoma Me180 and head and neck squamous cell carcinoma HEP2 cells at the transcriptional level. Cidofovir induced the accumulation of active p53 and pRb associated to induction of cyclin dependent kinase inhibitor p21WAF1/CIP1 in Me180 and HEP2 cells. p53 induction was also shown in Hela HPV-positive cervical carcinoma cell line. In addition, S phase cell cycle accumulation with concomitant decrease of cyclin A expression were associated to the antiproliferative activity of Cidofovir in HPV-treated cells. Combining Cidofovir to irradiation both in vivo and in nude mice xenografts resulted in a marked radiosensitization in HPV-positive cells, which was not observed in virus negative cells. This study provides the basis for a new anticancer strategy to enhance the antitumor effect of ionizing radiation in HPV-related cancers, without increase deleterious effects.

Alteration of the Sphingomyelin/Ceramide Pathway Is Associated with Resistance of Human Breast Carcinoma MCF7 Cells to Tumor Necrosis Factor-α-mediated Cytotoxicity

The interference of tumor necrosis factor-α (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-κB (NF-κB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.