Fathia Mami-Chouaib

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.

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.

Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the “missing self.” We show here that the Ig-like CD160 receptor expressed by circulating CD56dim+ NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94/CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160/HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

CD103 or LFA-1 Engagement at the Immune Synapse between Cytotoxic T Cells and Tumor Cells Promotes Maturation and Regulates T-cell Effector Functions

T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.

EMT impairs breast carcinoma cell susceptibility to CTL-mediated lysis through autophagy induction

Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.

Hypoxia-Dependent Inhibition of Tumor Cell Susceptibility to CTL-Mediated Lysis Involves NANOG Induction in Target Cells

Hypoxia is a major feature of the solid tumor microenvironment and is known to be associated with tumor progression and poor clinical outcome. Recently, we reported that hypoxia protects human non-small cell lung tumor cells from specific lysis by stabilizing hypoxia-inducible factor-1α and inducing STAT3 phosphorylation. In this study, we show that NANOG, a transcription factor associated with stem cell self renewal, is a new mediator of hypoxia-induced resistance to specific lysis. Our data indicate that under hypoxic conditions, NANOG is induced at both transcriptional and translational levels. Knockdown of the NANOG gene in hypoxic tumor cells is able to significantly attenuate hypoxia-induced tumor resistance to CTL-dependent killing. Such knockdown correlates with an increase of target cell death and an inhibition of hypoxia-induced delay of DNA replication in these cells. Interestingly, NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. To our knowledge, this study is the first to show that hypoxia-induced NANOG plays a critical role in tumor cell response to hypoxia and promotes tumor cell resistance to Ag-specific lysis.

Antitumor cytotoxic T‐lymphocyte response in human lung carcinoma: identification of a tumor‐associated antigen

Summary: We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a lung carcinoma of a patient with long survival. These clones showed a CD3+, CD8+, CD4–, CD28– phenotype and expressed a T‐cell receptor (TCR) encoded either by Vβ8‐Jβ1.5 or Vβ22‐Jβ1.4 rearrangements. Functional studies indicated that these clones mediated a high human leukocyte antigen (HLA)‐A2.1‐restricted cytotoxic activity against the autologous tumor cell line. Interestingly, TCRβ chain gene usage indicated that CTL clones identified in vitro were selectively expandedin vivo at the tumor site as compared to autologous peripheral blood lymphocytes (PBL). These findings provide evidence that an immune response may take place in non‐small cell lung carcinoma and that effector T cells may contribute to tumor regression. Further study indicated that the CTL clones recognized the same decamer peptide encoded by a mutated α‐actinin‐4 gene. Using tetramers of soluble HLA‐A2 molecules loaded with the mutated antigenic peptide, we have derived several anti‐α‐actinin‐4 T‐cell clones from patient PBL. These CTL, recognizing a truly tumor‐specific antigen, may play a role in the clinical evolution of this lung cancer patient. Adoptive transfer of CTL clones in a SCID/NOD mice model transplanted with autologous tumor supported their antitumor effect in vivo.

Analysis of T-cell-receptor β-chain-gene usage in peripheral-blood and tumor-infiltrating lymphocytes from human non-small-cell lung carcinomas

Non‐small‐cell lung cancers (NSCLC) are often infiltrated by T lymphocytes. It is postulated that the presence of tumor‐infiltrating lymphocytes (TIL) reflects a local host immune response against autologous tumors. To identify the nature of NSCLC TIL, we have characterized the molecular structure of the TCRβ chain expressed by infiltrating T cells and paired PBL from 9 untreated patients (4 LLC, 3 ADC and 2 SCC). For this purpose, we have used a high‐resolution PCR‐based method that determines CDR3 size patterns in TCRVβ sub‐families in fresh tumors and their corresponding autologous PBL samples. Oligoclonality in T‐cell populations was observed in 3 (Hor, Bla and Pub) out of 9 tumor biopsies analyzed. In contrast, the TCR repertoire of the 6 following patients as well as of all the autologous PBL was diverse, with virtually all Vβ specificities expressed. Among the 3 tumors with dominant T‐cell clonotypes, relative expansion of some T‐cell sub‐populations was observed. One patient (Hor) with significant TCRVβ21 expansion in tumor compared with autologous PBL, showed over‐expression of a particular TCRVβ chain with unique Vβ21‐D‐Jβ2.7 junctional region not detected in autologous PBL. TCRVβ21/Jβ2.7 expansion was also observed in IL‐2‐stimulated TIL cell lines and was confirmed by sequencing analysis of the V‐D‐J junctional region. These results strengthen the view that local antigen‐driven selection may occur, and support the hypothesis that anti‐tumor immune response may take place in some NSCLC. Int. J. Cancer 81:205–213, 1999.

Targeting WNT1-inducible signaling pathway protein 2 alters human breast cancer cell susceptibility to specific lysis through regulation of KLF-4 and miR-7 expression.

The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-β) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitionedxa0(EMT)xa0phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-β pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-β signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor.

Hypoxia-induced autophagy: a new player in cancer immunotherapy?

A major challenge in formulating an effective immunotherapy is to overcome the mechanisms of tumor escape from immunosurveillance. We showed that hypoxia-induced autophagy impairs cytotoxic T-lymphocyte (CTL)-mediated tumor cell lysis by regulating phospho-STAT3 in target cells. Autophagy inhibition in hypoxic cells decreases phospho-STAT3 and restores CTL-mediated tumor cell killing by a mechanism involving the ubiquitin proteasome system and SQSTM1/p62. Simultaneously boosting the CTL-response, using a TRP-peptide vaccination strategy, and targeting autophagy in hypoxic tumors, improves the efficacy of cancer vaccines and promotes tumor regression in vivo. Overall, in addition to its immunosuppressive effect, the hypoxic microenvironment also contributes to immunoresistance and can be detrimental to antitumor effector cell functions.