Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Tumor-host interactions Part 1: Stromal signatures of breast and prostate cancer Part 2: GLG1 and the control of the secretory pathway in tumor cells

Tumor-host interaction is a key determinant during cancer progression, from primary tumor growth to metastatic dissemination. At each step, tumor cells have to adapt to and subvert different types of microenvironment, leading to major phenotypic and genotypic alterations that affect both tumor and surrounding stromal compartments. Understanding the molecular mechanisms that govern tumor-host interplay may be essential for better comprehension of tumorigenesis in an effort to improve current anti-cancer therapies. The present work is composed of two projects that address tumor-host interactions from two different perspectives, the first focusing on the characterization of tumor-associated stroma and the second on membrane trafficking in tumor cells. Part 1. To selectively address stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to analyze the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Comparison showed that invasive breast and prostate cancer elicit distinct, tumor-specific stromal responses, with a limited panel of shared induced and/or repressed genes. Both breast and prostate tumor-specific deregulated stromal gene sets displayed statistically significant survival-predictive ability for their respective tumor type. By contrast, a stromal gene signature common to both tumor types did not display prognostic value, although expression of two individual genes within this common signature was found to be associated with patient survival. Part 2. GLG1 is known as an E-selectin ligand and an intracellular FGF receptor, depending on cell type and context. Immunohistochemical and immunofluorescence analyses showed that GLG1 is primarily localized in the Golgi of human tumor cells, a central location in the biosynthetic/secretory pathways. GLG1 has been shown to interact with and to recruit the ARF GEF BIGI to the Golgi membrane. Depletion of GLG1 or BIGI markedly reduced ARF3 membrane localization and activation, and altered the Golgi structure. Interestingly, these perturbations did not impair constitutive secretion in general, but rather seemed to impair secretion of a specific subset of proteins that includes MMP-9. Thus, GLG1 coordinates ARF3 activation by recruiting BIGI to the Golgi membrane, thereby affecting secretion of specific molecules. - Les interactions tumeur-hôte constituent un élément essentiel à la progression tumorale, de la croissance de la tumeur primaire à la dissémination des métastases. A chaque étape, les cellules tumorales doivent s'adapter à différents types de microenvironnement et les détourner à leur propre avantage, donnant lieu à des altérations phénotypiques et génotypiques majeures qui affectent aussi bien la tumeur elle-même que le compartiment stromal environnant. L'étude des mécanismes moléculaires qui régissent les interactions tumeur-hôte constitue une étape essentielle pour une meilleure compréhension du processus de tumorigenèse dans le but d'améliorer les thérapies anti cancer existantes. Le travail présenté ici est composé de deux projets qui abordent la problématique des interactions tumeur-hôte selon différentes perspectives, le premier se concentrant sur la caractérisation du stroma tumoral et le second sur le trafic intracellulaire des cellules tumorales. Partie 1. Pour examiner les changements d'expression des gènes dans le stroma en réponse à la progression du cancer, des puces à ADN Affymetrix ont été utilisées afin d'analyser les transcriptomes des cellules stromales issues de carcinomes invasifs du sein et de la prostate et collectées par microdissection au laser. L'analyse comparative a montré que les cancers invasifs du sein et de la prostate provoquent des réponses stromales spécifiques à chaque type de tumeur, et présentent peu de gènes induits ou réprimés de façon similaire. L'ensemble des gènes dérégulés dans le stroma associé au cancer du sein, ou à celui de la prostate, présente une valeur pronostique pour les patients atteints d'un cancer du sein, respectivement de la prostate. En revanche, la signature stromale commune aux deux types de cancer n'a aucune valeur prédictive, malgré le fait que l'expression de deux gènes présents dans cette liste soit liée à la survie des patients. Partie 2. GLG1 est connu comme un ligand des sélectines E ainsi que comme récepteur intracellulaire pour des facteurs de croissances FGFs selon le type de cellule dans lequel il est exprimé. Des analyses immunohistochimiques et d'immunofluorescence ont montré que dans les cellules tumorales, GLG1 est principalement localisé au niveau de l'appareil de Golgi, une place centrale dans la voie biosynthétique et sécrétoire. Nous avons montré que GLG1 interagit avec la protéine BIGI et participe à son recrutement à la membrane du Golgi. L'absence de GLG1 ou de BIGI réduit drastiquement le pool d'ARF3 associé aux membranes ainsi que la quantité d'ARF3 activés, et modifie la structure de l'appareil de Golgi. Il est particulièrement intéressant de constater que ces perturbations n'ont pas d'effet sur la sécrétion constitutive en général, mais semblent plutôt affecter la sécrétion spécifique d'un sous-groupe défini de protéines comprenant MMP-9. GLG1 coordonne donc l'activation de ARF3 en recrutant BIGI à la membrane du Golgi, agissant par ce moyen sur la sécrétion de molécules spécifiques.

Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317-326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317-326 sequence of RasGAP (TAT-RasGAP₃₁₇₋₃₂₆), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP₃₁₇₋₃₂₆ did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP₃₁₇₋₃₂₆ was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.

Can hTERT peptide (540-548) -specific CD8 T cells recognize and kill tumor cells?

This commentary reviews the data on HLA-A2-restricted CD8 T cells specific for peptide (540-548) derived from hTERT (human telomerase reverse transcriptase). Several studies have reported the successful generation of such T cells (1, 2, 3). However, tumor recognition was observed in some, but not all, studies. More data are required to elucidate whether hTERT peptide (540-548) -specific T cells can indeed recognize and destroy tumor cells. It would be highly useful if telomerase would emerge as a universal tumor antigen that can be targeted in the cancer immunotherapy of HLA-A2 positive patients.

Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b(+) myelomonocytic cells.

It is well established that cancer cells can recruit CD11b(+) myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor-infiltrating CD11b(+) myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564-1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b(+) myeloid cells isolated from peripheral blood and from tumors, using six-color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor-derived samples concur to generate artifacts in the phenotype of tumor-infiltrating CD11b(+) subpopulations. We show that the heterogeneity of tumor-infiltrating CD11b(+) subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor-infiltrating CD11b(+) cells was particularly sensitive to these parameters compared to circulating CD11b(+) cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor-infiltrating CD11b(+) cell populations.

T Cells Bearing a Chimeric Antigen Receptor against Prostate-Specific Membrane Antigen Mediate Vascular Disruption and Result in Tumor Regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

Involvement of circulating CEA in liver metastases from colorectal cancers re-examined in a new experimental model.

Both experimental and clinical data show evidence of a correlation between elevated blood levels of carcinoembryonic antigen (CEA) and the development of liver metastases from colorectal carcinomas. However, a cause-effect relationship between these two observations has not been demonstrated. For this reason, we developed a new experimental model to evaluate the possible role of circulating CEA in the facilitation of liver metastases. A CEA-negative subclone from the human colon carcinoma cell line CO115 was transfected either with CEA-cDNA truncated at its 3' end by the deletion of 78 base pairs leading to the synthesis of a secreted form of CEA or with a full-length CEA-cDNA leading to the synthesis of the entire CEA molecule linked to the cell surface by a GPI anchor. Transfectants were selected either for their high CEA secretion (clone CO115-2C2 secreting up to 13 microg CEA per 10(6) cells within 72 h) or for their high CEA membrane expression (clone CO115-5F12 expressing up to 1 x 10(6) CEA molecules per cell). When grafted subcutaneously, CO115-2C2 cells gave rise to circulating CEA levels that were directly related to the tumour volume (from 100 to 1000 ng ml(-1) for tumours ranging from 100 to 1000 mm3), whereas no circulating CEA was detectable in CO115 and CO115-5F12 tumour-bearing mice. Three series of nude mice bearing a subcutaneous xenograft from either clone CO115-2C2 or the CO115-5F12 transfectant, or an untransfected CO115 xenograft, were further challenged for induction of experimental liver metastases by intrasplenic injection of three different CEA-expressing human colorectal carcinoma cell lines (LoVo, LS174T or CO112). The number and size of the liver metastases were shown to be independent of the circulating CEA levels induced by the subcutaneous CEA secreting clone (CO115-2C2), but they were directly related to the metastatic properties of the intrasplenically injected tumour cells.

Characterization of tumor antigen specific CD8+ T cells and semi-invariant Vα24/Vβ11 NKT cells in tumor invaded liver

Summary: Detailed knowledge on tumor antigen expression and specific immune cells is required for a rational design of immunotherapy for patients with tumor invaded liver. In this study, we confirmed that Cancer/Testis (CT) tumor-associated antigens are frequently expressed in hepatocellular carcinoma (HCC) and searched for the presence of CD8+ T cells specific for these antigens. In 2/10 HLA-A2+ patients with HCC, we found that MAGE-A10 and/or SSX-2 specific CD8+ T cells naturally responded to the disease, since they were enriched in tumor lesions but not in non-tumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, suggesting that these CTL were selected in vivo for high avidity antigen recognition, providing the rational for specific immunotherapy of HCC, based on immunization with CT antigens such as MAGE-Al 0 and SSX-2. Type 1 NKT cells express an invariant TCR α chain (Vα24.1α18, paired with Vβ11 in human) and share a specific reactivity to αGalactosylceramide (αGC) presented by CD1d. These cells can display paradoxical immuno-regulatory properties including strong anti-tumor effects upon αGC administration in murine models. To understand why NKT cells were not sufficiently protective against tumor development in patients with tumor invaded liver, we characterized the diversity of Vα24/Vβ11 NKT cells in healthy donors (HD) and cancer patients: NKT cells from HD and patients were generally diverse in terms of TCR β chain (Vβ11) variability and NKT cells from HD showed a variable recognition of αGC loaded CD 1 d multimers. Vα24/ Vβ11 NKT cells can be divided in 3 populations, the CD4, DN (CD4-/CD8-) and CD8 NKT cell subsets that show distinct ability of cytokine production. In addition, our functional analysis revealed that DN and CD8 subsets displayed a higher cytolytic potential and a weaker IFNγ release than the CD4 NKT cell subset. NKT cell subsets were variably represented in the blood of HD and cancer patients. However, HD with high NKT cell frequencies displayed an enrichment of the DN and CD8 subsets, and few of them were suggestive of an oligoclonal expansion in vivo. Comparable NKT cell frequencies were found between blood, non-tumoral liver and tumor of patients. In contrast, we identified a gradual enrichment of CD4 NKT cells from blood to the liver and to the tumor, together with a decrease of DN and CD8 NKT cell subsets. Most patient derived NKT cells were unresponsive upon αGalactosylceramide stimulation ex vivo; NKT cells from few patients displayed a weak responsiveness with different cytokine polarization. The NKT cell repertoire was thus different in tumor tissue, suggesting that CD4 NKT cells infiltrating tumors may be detrimental for protection against tumors and instead may favour the tumor growth/recurrence as recently reported in mice. Résumé en français scientifique : Afin de développer le traitement des patients porteurs d'une tumeur dans le foie par immunothérapie, de nouvelles connaissances sont requises concernant l'expression d'antigènes par les tumeurs et les cellules immunitaires spécifiques de ces antigènes. Nous avons vérifié que des antigènes associés aux tumeurs, tels que les antigènes « Cancer-Testis » (CT), sont fréquemment exprimés par le carcinome hepatocéllulaire (CHC). La recherche de lymphocytes T CD8+ spécifiques (CTL) de ces antigènes a révélé que des CTL spécifiques de MAGE-A10 et/ou SSX-2 ont répondu naturellement à la tumeur chez 2/10 patients étudiés. Ces cellules étaient présentes dans les lésions tumorales mais pas dans le foie adjacent. De plus, ces CTL ont démontré une activité cytolytique forte et spécifique contre les cellules tumorales in vitro, ce qui suggère que ces CTL ont été sélectionnés pour une haute avidité de reconnaissance de l'antigène in vivo. Ces données fournissent une base pour l'immunothérapie spécifique du CHC, en proposant de cibler les antigènes CT tels que MAGE-A10 ou SSX-2. Les cellules NKT de type 1 ont une chaîne α de TCR qui est invariante (chez l'homme, Vα24Jα18, apparié avec Vβ11) et reconnaissent spécifiquement l'αGalactosylceramide (αGC) présenté par CD1d. Ces cellules ont des propriétés immuno¬régulatrices qui peuvent être parfois contradictoires et leur activation par l'αGC induit une forte protection anti-tumorale chez la souris: Afin de comprendre pourquoi ces cellules ne sont pas assez protectrices contre le développement des tumeurs dans le foie chez l'homme, nous avons étudié la diversité des cellules NKT Vα24/Vβ11 d'individus sains (IS) et de patients cancéreux. Les cellules NKT peuvent être sous-divisées en 3 populations : Les CD4, DN (CD4- /CD8-) ou CDS, qui ont la capacité de produire des cytokines différentes. Nos analyses fonctionnelles ont aussi révélé que les sous-populations DN et CD8 ont un potentiel cytolytique plus élevé et une production d'IFNγ plus faible que la sous-population CD4. Ces sous-populations sont représentées de manière variable dans le sang des IS ou des patients. Cependant, les IS avec un taux élevé de cellules NKT ont un enrichissement des sous- populations DN ou CDS, et certains suggèrent qu'il s'agit d'une expansion oligo-clonale in vivo. Les patients avaient des fréquences comparables de cellules NKT entre le sang, le foie et la tumeur. Par contre, la sous-population CD4 était progressivement enrichie du sang vers le foie et la tumeur, tandis que les sous-populations DN ou CD8 était perdues. La plupart des cellules NKT des patients ne réagissaient pas lors de stimulation avec l'αGC ex vivo et les cellules NKT de quelques patients répondaient faiblement et avec des polarisations de cytokines différentes. Ces données suggèrent que les cellules NKT CD4, prédominantes dans les tumeurs, sont inefficaces pour la lutte anti-tumorale et pourraient même favoriser la croissance ou la récurrence tumorale. Donc, une mobilisation spécifique des cellules NKT CD4 négatives par immunothérapie pourrait favoriser l'immunité contre des tumeurs chez l'homme. Résumé en français pour un large public Au sein des globules blancs, les lymphocytes T expriment un récepteur (le TCR), qui est propre à chacun d'entre eux et leur permet d'accrocher de manière très spécifique une molécule appelée antigène. Ce TCR est employé par les lymphocytes pour inspecter les antigènes associés avec des molécules présentatrices à la surface des autres cellules. Les lymphocytes T CD8 reconnaissent un fragment de protéine (ou peptide), qui est présenté par une des molécules du Complexe Majeur d'Histocompatibilité de classe I et tuent la cellule qui présente ce peptide. Ils sont ainsi bien adaptés pour éliminer les cellules qui présentent un peptide issu d'un virus quand la cellule est infectée. D'autres cellules T CD8 reconnaissent des peptides comme les antigènes CT, qui sont produits anormalement par les cellules cancéreuses. Nous avons confirmé que les antigènes CT sont fréquemment exprimés par le cancer du foie. Nous avons également identifié des cellules T CD8 spécifiques d'antigènes CT dans la tumeur, mais pas dans le foie normal de 2 patients sur 10. Cela signifie que ces lymphocytes peuvent être naturellement activés contre la tumeur et sont capables de la trouver. De plus les lymphocytes issus d'un patient ont démontré une forte sensibilité pour reconnaître l'antigène et tuent spécifiquement les cellules tumorales. Les antigènes CT représentent donc des cibles intéressantes qui pourront être intégrés dans des vaccins thérapeutiques du cancer du foie. De cette manière, les cellules T CD8 du patient lui-même pourront être induites à détruire de manière spécifique les cellules cancéreuses. Un nouveau type de lymphocytes T a été récemment découvert: les lymphocytes NKT. Quand ils reconnaissent un glycolipide présenté par la molécule CD1d, ils sont capables, de manière encore incomprise, d'initier, d'augmenter, ou à l'inverse d'inhiber la défense immunitaire. Ces cellules NKT ont démontré qu'elles jouent un rôle important dans la défense contre les tumeurs et particulièrement dans le foie des souris. Nous avons étudié les cellules NKT de patients atteints d'une tumeur dans le foie, afin de comprendre pourquoi elles ne sont pas assez protectrice chez l'homme. Les lymphocytes NKT peuvent être sous-divisés en 3 populations: Les CD4, les DN (CD4-/CD8-) et les CD8. Ces 3 classes de NKT peuvent produire différents signaux chimiques appelés cytokines. Contrairement aux cellules NKT DN ou CDS, seules les cellules NKT CD4 sont capables de produire des cytokines qui sont défavorables pour la défense anti-tumorale. Par ailleurs nous avons trouvé que les cellules NKT CD4 tuent moins bien les cellules cancéreuses que les cellules NKT DN ou CD8. L'analyse des cellules NKT, fraîchement extraites du sang, du foie et de la tumeur de patients a révélé que les cellules NKT CD4 sont progressivement enrichies du sang vers le foie et la tumeur. La large prédominance des NKT CD4 à l'intérieur des tumeurs suggère que, chez l'homme, ces cellules sont inappropriées pour la lutte anti-tumorale. Par ailleurs, la plupart des cellules NKT de patients n'étaient pas capables de produire des cytokines après stimulation avec un antigène. Cela explique également pourquoi ces cellules ne protègent pas contre les tumeurs dans le foie.

Melan-A-specific cytotoxic T cells are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4.

PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Résumé Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mésenchymateuses modifiées génétiquement afin de surexprimer localement les deux chémokines CXCL13 et CCL2 ensemble ou chacune séparément à l'intérieur d'une tumeur solide. CXCL13 est supposé induire des structures lymphoïdes ectopiques. Un niveau élevé de CCL2 est présumé initier une inflammation aiguë. La combinaison des deux effets amène à un nouveau modèle d'étude des mécanismes régulateur de la tolérance périphérique et de l'immunité tumorale. Les connaissances acquises grâce à ce modèle pourraient permettre le développement ou l'amélioration des thérapies immunes du cancer. Le but premier de ce travail a été l'établissement d'un modèle génétique de la souris permettant d'exprimer spécifiquement dans la tumeur les deux chémokines d'intérêt à des niveaux élevés. Pour accomplir cette tâche, qui est en fait une thérapie génétique de tumeurs solides, deux types de cellules porteuses potentielles ont été évaluées. Des cellules CD8+ T et des cellules mésenchymateuses de la moelle osseuse transférées dans des receveurs portant une tumeur. Si on pouvait répondre aux besoins de la thérapie génétique, indépendamment de la thérapie immune envisagée, on posséderait là un outil précieux pour bien d'autres approches thérapeutiques. Plusieurs lignées de souris transgéniques ont été générées comme source de cellules CD8+ T modifiées afin d'exprimer les chémokines d'intérêt. Dans une approche doublement transgénique les propriétés de deux promoteurs spécifiques de cellules T ont été combinées en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confère une dépendance d'activation et le promoteur distal de lck assure une forte expression constitutive dès que les cellules CD8+ T ont été activées. Les transgènes construits ont montré une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activées ont été obtenues. Ces cellules peuvent maintenant être utilisées avec différents protocoles pour transférer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'évaluer leur capacité en tant que porteuse de chémokine d'infiltrer la tumeur. L'établissement de souris transgéniques, qui expriment pareillement CXCL13 est prévu dans un avenir proche. L'évaluation de cellules mésenchymateuses de la moelle osseuse a démontré que ces cellules se greffent efficacement dans le stroma tumoral suite à la co-injection avec des cellules tumorales. Cela représente un outil précieux pour la recherche, vu qu'il permet d'introduire des cellules manipulées dans un modèle tumoral. Les résultats confirment partiellement d'autres résultats rapportés dans un modèle amélioré. Cependant, l'efficacité et la spécificité suggérées de la migration systémique de cellules mésenchymateuses de la moelle osseuse dans une tumeur n'ont pas été observées dans notre modèle, ce qui indique, que ces cellules ne se prêtent pas à une utilisation thérapeutique. Un autre résultat majeur de ce travail est l'établissement de cultures de cellules mésenchymateuses de la moelle osseuse in vitro conditionnées par des tumeurs, ce qui a permis à ces cellules de s'étendre plus rapidement en gardant leur capacité de migration et de greffe. Cela offre un autre outil précieux, vu que la culture in vitro est un pas nécessaire pour une manipulation thérapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.

Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein.

Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.