Guillaume Dorothée

MyD88 and Type I Interferon Receptor-Mediated Chemokine Induction and Monocyte Recruitment during Listeria monocytogenes Infection

Monocytes play a central role in defense against infection, but the mechanisms promoting monocyte recruitment and activation remain incompletely defined. Defense against Listeria monocytogenes, an intracellular bacterial pathogen, requires in vivo MCP-1 induction and CCR2-dependent recruitment of Ly6Chigh monocytes from bone marrow to sites of infection. Herein, we demonstrate that infection of bone marrow-derived macrophages with virulent L. monocytogenes induces MCP-1 expression in two phases. The first phase is rapid, induces low-level production of MCP-1, and is dependent on TLR/MyD88 signaling. The second phase promotes prolonged, higher level MCP-1 secretion and is dependent on signaling via the type I IFN receptor (IFNAR). Although attenuated L. monocytogenes strains that remain confined to the phagosome trigger TLR/MyD88-mediated signals and induce low-level MCP-1 expression, only cytosol-invasive bacteria promote IFNAR-dependent MCP-1 expression. In vivo, deficiency of either MyD88 or IFNAR signaling does not impair early monocyte emigration from bone marrow and recruitment to infected spleen. Loss of both MyD88 and IFNAR-mediated MCP-1 induction, however, results in deficient Ly6Chigh monocyte recruitment and increased susceptibility to L. monocytogenes infection. Our studies demonstrate that distinct but partially overlapping signal transduction pathways provide redundancy that ensures optimal monocyte recruitment to sites of microbial infection.

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.

Mutant |[alpha]|-actinin-4 promotes tumorigenicity and regulates cell motility of a human lung carcinoma

The precise role of α-actinin-4 encoding gene (ACTN4) is not very well understood. It has been reported to elicit tumor suppressor activity and to regulate cellular motility. To further assess the function of human ACTN4, we studied a lung carcinoma cell line expressing a mutated α-actinin-4, which is recognized as a tumor antigen by autologous CD8+ cytotoxic T lymphocytes (CTL). Confocal immunofluorescence microscopy indicated that, while wild-type (WT) α-actinin-4 stains into actin cytoskeleton and cell surface ruffles, the mutated protein is only dispersed in the cytoplasm of the lung carcinoma cells. This loss of association with the cell surface did not appear to correlate with a decrease in in vitro α-actinin-4 crosslinking to filamentous (F)-actin. Interestingly, experiments using cell lines stably expressing ACTN4 demonstrated that as opposed to WT gene, mutant ACTN4 was unable to inhibit tumor cell growth in vitro and in vivo. Moreover, the expression of mutant α-actinin-4 resulted in the loss of tumor cell capacity to migrate. The identification of an inactivating mutation in ACTN4 emphasizes its role as a tumor suppressor gene and underlines the involvement of cytoskeleton alteration in tumor development and metastasis.

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.

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.

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.

Wild-type p53 induced sensitization of mutant p53 TNF-resistant cells: Role of caspase-8 and mitochondria

In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome c to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the cross-talk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.

MHC-Independent Genetic Factors Control the Magnitude of CD4+ T Cell Responses to Amyloid-β Peptide in Mice through Regulatory T Cell-Mediated Inhibition

Accumulation of amyloid-β peptide (Aβ) is considered the triggering factor of pathogenic lesions in Alzheimer’s disease (AD), and vaccines targeting Aβ are promising therapeutic options. However, the occurrence of meningoencephalitides attributed to T cell responses in 6% of Aβ-immunized patients underscores the need for a better understanding of T cell responses to Aβ. We characterized the parameters controlling the magnitude of Aβ-specific CD4+ T cell responses in mice. T cell responsiveness to Aβ1-42 was highly heterogeneous between mouse strains of different H-2 haplotypes, with SJL/J (H-2s) mice displaying a strong response, mainly specific for Aβ10-24, and C57BL/6 (H-2b) mice displaying a weak response to Aβ16-30. Surprisingly, C57BL/6 mice congenic for the H-2s haplotype (B6.H-2S), which display a “permissive” MHC class II allele for presentation of the immunodominant Aβ10-24 epitope, showed a very weak CD4+ T cell response to Aβ, suggesting that MHC-independent genes downmodulate Aβ-specific CD4+ T cell responses in C57BL/6 background. Vaccine-induced CD4+ T cell responses to Aβ were significantly enhanced in both C57BL/6 and B6.H-2S mice upon depletion of regulatory T cells (Tregs), whereas Treg-depleted SJL/J mice displayed unaltered Aβ-specific T cell responses. Finally, Treg depletion in C57BL/6 transgenic APPPS1 mice, a mouse model of AD, results in enhanced vaccine-induced CD4+ T cell responses in AD compared with wild-type animals. We concluded that the magnitude of Aβ-specific CD4+ T cell responses is critically controlled in both physiological and pathological settings by MHC-independent genetic factors that determine the overall potency of Aβ-specific Treg responses.