Faten El Hage

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO2) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1α (HIF-1α) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1α and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1α inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1α resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phopho-STAT3 and HIF-1α are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1α in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

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.

In Situ Sensory Adaptation of Tumor-Infiltrating T Lymphocytes to Peptide-MHC Levels Elicits Strong Antitumor Reactivity

We have isolated from tumor-infiltrating lymphocytes (TIL) and PBL of a lung carcinoma patient several tumor-specific T cell clones displaying similar peptide-MHC tetramer staining and expressing a unique TCR. Although these clones elicited identical functional avidity and similar cytolytic potential, only T cell clones derived from TIL efficiently lysed autologous tumor cells. Interestingly, all of these clones expressed the same T cell surface markers except for the TCR inhibitory molecule CD5, which was expressed at much lower levels in TIL than in PBL. Video-imaging recordings demonstrated that, although both T cell clones could form stable conjugates with tumor cells, the Ca2+ response occurred in TIL clones only. Significantly, analysis of a panel of circulating clones indicated that antitumor cytolytic activity was inversely proportional to CD5 expression levels. Importantly, CD5 levels in TIL appeared to parallel the signaling intensity of the TCR/peptide-MHC interaction. Thus, in situ regulation of CD5 expression may be a strategy used by CTL to adapt their sensitivity to intratumoral peptide-MHC levels.

Human CD5 Protects Circulating Tumor Antigen-Specific CTL from Tumor-Mediated Activation-Induced Cell Death

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.

Different Expression Levels of the TAP Peptide Transporter Lead to Recognition of Different Antigenic Peptides by Tumor-Specific CTL

Decreased antigenicity of cancer cells is a major problem in tumor immunology. This is often acquired by an expression defect in the TAP. However, it has been reported that certain murine Ags appear on the target cell surface upon impairment of TAP expression. In this study, we identified a human CTL epitope belonging to this Ag category. This epitope is derived from preprocalcitonin (ppCT) signal peptide and is generated within the endoplasmic reticulum by signal peptidase and signal peptide peptidase. Lung cancer cells bearing this antigenic peptide displayed low levels of TAP, but restoration of their expression by IFN-γ treatment or TAP1 and TAP2 gene transfer abrogated ppCT Ag presentation. In contrast, TAP upregulation in the same tumor cells increased their recognition by proteasome/TAP-dependent peptide-specific CTLs. Thus, to our knowledge, ppCT16–25 is the first human tumor epitope whose surface expression requires loss or downregulation of TAP. Lung tumors frequently display low levels of TAP molecules and might thus be ignored by the immune system. Our results suggest that emerging signal peptidase-generated peptides represent alternative T cell targets, which permit CTLs to destroy TAP-impaired tumors and thus overcome tumor escape from CD8+ T cell immunity.

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.

TAP expression level in tumor cells defines the nature and processing of MHC class I peptides for recognition by tumor‐specific cytotoxic T lymphocytes

We identified that the antigen preprocalcitonin (ppCT) is recognized on a human lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor‐infiltrating lymphocytes. The antigenic peptide ppCT16–25 is encoded by the gene calcitonin‐related polypeptide alpha (CALCA), which codes for CT and is overexpressed in several lung carcinomas compared with normal tissues. The ppCT peptide is derived from the C‐terminal region of the signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing (TAP)1/TAP2 heterodimeric complexes. Instead, processing occurs within the endoplasmic reticulum by a novel mechanism involving signal pepsidase (SP) and signal peptide peptidase (SPP). Although lung cancer cells bearing the ppCT16–25 epitope displayed low levels of TAP, restoration of TAP expression by interferon (IFN)‐γ treatment or by TAP1/TAP2 gene transfer inhibited ppCT antigen presentation. Thus, the ppCT16–25 human tumor epitope requires low TAP expression for efficient presentation. These results indicate that emerging SP‐generated peptides represent alternative T cell targets that permit cytotoxic T lymphocytes to destroy TAP‐impaired tumors, a process that helps to overcome tumor escape from CD8+ T cell immunity. Additionally, our data suggest that ppCT is a promising candidate for cancer immunotherapy.

Réponse immune et cancer

Cellular transformation is initiated by genetic and epigenetic mutations that activate oncogenes and inactivate tumor suppressor pathways. Cancers thus arise when somatic cells escape intrinsic and extrinsic tumor suppressor mechanisms in the context of their cellular microenvironment. Given the well established importance of the immune system at controlling and shaping developing tumors, pointing the different strategies of tumor escape may provide important insights for the development of effective cancer therapies. In respect, a better understanding of the molecular interactions between tumors and the host immune system may thus allow the development of novel integrated approaches based on the simultaneous control of tumor escape pathways and the activation of anti-cancer immune responses. We hereafter review the currently known escape strategies developed by tumors and discuss the very limited success of trials using active immunization with vaccines or adoptive immunotherapy conducted to date, with a focus on potential therapeutic avenues. We believe that the induction of clinically relevant anti-cancer immunity and tumor rejection require an orchestrated set of events that are thus far impossible to activate by a single approach. Therefore, combining immunotherapy with conventional therapies may help in breaking down the existing barriers.