Hamid Echchakir

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.

Preprocalcitonin signal peptide generates a cytotoxic T lymphocyte-defined tumor epitope processed by a proteasome-independent pathway

We identified an antigen recognized on a human non-small-cell lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide is presented by HLA-A2 and is encoded by the CALCA gene, which codes for calcitonin and for the α-calcitonin gene-related peptide. The peptide is derived from the carboxy-terminal region of the preprocalcitonin signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing. Processing occurs within the endoplasmic reticulum of all tumoral and normal cells tested, including dendritic cells, and it involves signal peptidase and the aspartic protease, signal peptide peptidase. The CALCA gene is overexpressed in medullary thyroid carcinomas and in several lung carcinomas compared with normal tissues, leading to recognition by the T cell clone. This new epitope is, therefore, a promising candidate for cancer immunotherapy.

Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution

We have previously identified an antigen (Ag) recognized on a human large cell carcinoma of the lung by a tumor-specific cytotoxic T lymphocyte clone derived from autologous tumor infiltrating lymphocytes (TILs). The antigenic peptide is presented by HLA-A2 molecules and is encoded by a mutated α-actinin-4 (ACTN4) gene. In the present report, we have isolated two anti-α-actinin-4 T cell clones from the same patient TIL and from his peripheral blood lymphocytes (PBLs) by using tetramers of soluble HLA-A2 molecules loaded with the mutated peptide. Although all of the clones displayed similar tetramer labeling, those isolated from PBL showed lower avidity of Ag recognition and killed the specific target much less efficiently, indicating that tetramer staining does not correlate with clone avidity/tumor reactivity. T cell receptor (TCR) analysis revealed that α-actinin-4-reactive clones used distinct α and β chain rearrangements, demonstrating TCR repertoire diversity. Interestingly, TCRβ chain gene usage indicated that only Ag-specific clones with high functional avidity were expanded at the tumor site, whereas a low-avidity clone was exclusively amplified in patient peripheral blood. Our results point to the existence of distinct but overlapping antitumor TCR repertoires in TIL and PBL and suggest a selective in situ expansion of tumor-specific cytotoxic T lymphocyte with high avidity/tumor reactivity.

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.

Functional and molecular characterization of a KIR3DL2/p140 expressing tumor-specific cytotoxic T lymphocyte clone infiltrating a human lung carcinoma

T lymphocytes infiltrating a human lung carcinoma stimulated in vitro with autologous tumor cell line showed a TCRVβ13.6+ T-cell expansion. This subset was isolated using TCRVβ-specific antibody and several T-cell clones were generated. All these clones expressed a unique Vβ13.6-Jβ2.7 TCR with the same junctional region strongly suggesting that they derived from the same cell. They were CD8+/CD28− and expressed the MHC class I binding killer cell Ig-like receptor (KIR)3DL2/p140, but not KIR3DL1/p70, KIR2DL1/p58.1 and KIR2DL2/3/p58.2. Sequence analysis indicated that KIR3DL2/p140 cDNA was identical to the previously reported 3DL2*002 allele except for two nucleic acid substitutions. Functional studies showed that KIR3DL2/p140+ CTL secrete a significant level of IFNγ and mediate an HLA-A2-restricted cytotoxicity against the autologous and some allogeneic tumor cells but not towards the autologous EBV-B cells. Strikingly, both the lytic and the cytokine secretion activities induced upon specific cell interactions were unaffected by anti-KIR3DL2/p140 antibody. In addition, crosslinking KIR3DL2/p140 molecules on CTL did not result into the modification of cytotoxicity and cytokine production triggered by anti-CD3 antibody. These results strongly suggest that, as opposed to distinct KIR expressed by CTL, the in vitro KIR3DL2/p140 engagement does not result into inhibitory (nor activatory) effects on tumor-specific CTL.

Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte–induced apoptosis of autologous human lung-carcinoma cells

We have isolated a cytotoxic T lymphocyte (CTL) clone, Heu161, that reacts specifically with the human autologous lung carcinoma cell line IGR‐Heu. We first demonstrated that IGR‐Heu lacked Fas‐receptor expression and was resistant to CD95‐induced apoptosis. To further elucidate the role of Fas in tumor immune surveillance, we have stably transfected IGR‐Heu with a Fas‐expression vector and isolated CD95‐sensitive and ‐resistant clones. Our data indicated that the resistance of 2 selected Fas‐transfected clones to CD95‐mediated lysis correlated with down‐regulation of caspase‐8 or its lack of cleavage and subsequent activation. All Fas transfectants, either sensitive or resistant to anti‐Fas agonistic antibody, were as efficiently lysed by the CTL clone as the parental cell line. In addition, neither anti‐Fas‐blocking antibody nor Fas‐Fc molecule inhibited T‐cell lysis of Fas‐sensitive tumor clone. This cytotoxicity was extracellular Ca2+‐dependent and abolished in the presence of EGTA, indicating that it was mainly granzyme‐mediated. Interestingly, although the caspase inhibitor z‐VAD‐fmk had no effect on tumor‐cell lysis, it efficiently blocked target DNA damage triggered by autologous CTLs via the granule exocytosis pathway, indicating that the latter event was caspase‐dependent. The present results suggest that lung carcinoma‐specific CTLs use mainly a granule exocytosis‐dependent pathway to lyse autologous target cells and that these effectors are able to circumvent alteration of the Fas‐triggered intracellular signalling pathway via activation of a caspase‐independent cytoplasmic death mechanism.

Role of p53 in the sensitization of tumor cells to apoptotic cell death

Immunotherapy of cancer has always represented a very attractive fourth-modality therapeutic approach. Over the past few years, advances in the identification of tumor antigens have opened new perspectives and provided new opportunities for a more accurate immunotherapy of cancer. However, when applied to patients with established tumors, it rarely leads to an objective response. This is in part due to the fact that tumors evade host immunity at both the induction and effector phases. In this regard, several different functional defects in T-lymphocytes that infiltrate cancers have been reported. Indeed, lymphocytes of patients with advanced malignancies are hyporeactive and functionally compromised. Furthermore, it has become clear that immunotherapeutic and gene therapeutic approaches aimed at the induction of anti-tumor cytotoxic responses should consider the resistance of tumor cells to cytotoxic mechanisms. Thus, understanding of tumor escape mechanisms may be the key to a successful immunotherapy for cancer. How tumors escape immunological destruction following the acquisition of resistance to cell death and the potential role the tumor suppressor p53 protein in immunosensitization of tumor cells will be discussed.