Caractérisation de DKK1 comme antigène tumoral et manipulation des lymphocytes T CD8 : utilisation de la voie de Wnt en immunothérapie du cancer

L’immunothérapie tumorale à médiation cellulaire est un traitement qui utilise le système immunitaire des patients afin d’induire une réponse des lymphocytes T CD8+ (T CD8+) contre la tumeur. Cette réponse est produite suite à la reconnaissance des antigènes par les T CD8+. Ces cibles sont appelées antigènes tumoraux (TAA) et définies comme des protéines exprimées par les cellules cancéreuses mais absentes des tissus normaux. Par une approche bio-informatique, notre laboratoire a identifié Dickkopf-1 (DKK1), une protéine inhibitrice de la voie de Wnt, comme un TAA potentiel. Une immunothérapie à médiation cellulaire efficace requiert l’identification de TAA candidats pertinents. Le traitement de patients par immunothérapie pourrait également être améliorées par l’augmentation de la puissance d’action anti-tumorale ainsi que la persistante des T CD8+ spécifiques aux TAA. Ce projet de doctorat se divise en deux parties : 1- La caractérisation de l’expression de DKK1 dans les cancers communs et la détermination de son immunogénicité afin de valider sa candidature comme TAA. 2- La reprogrammation des T CD8+, de patients atteints d’un cancer commun, vers un phénotype moins différentié afin d’augmenter leur potentiel anti-tumoral et leur persistance. Dans le premier objectif, nous avons caractérisé l’expression de DKK1 dans le cancer du sein et dans d’autres cancers communs. Le profil d’expression de DKK1 a été étudié par RT-PCR et par ELISA dans plusieurs lignées cellulaires de cancer et dans les tissus normaux. L’expression de DKK1 a aussi été étudiée dans des échantillons cliniques provenant de cancers du sein, du poumon et du rein. Trente pourcents (30%) des tumeurs provenant d’un cancer du sein exprimaient DKK1. La moitié des tumeurs DKK1(+) était triple négative, donc pas de récepteurs d’œstrogène et de progestérone et était Her-2/neu(-) (ces patientes ont des possibilités de traitements très restreintes). De plus, 50% des échantillons cliniques de tumeurs du poumon et 30% des tumeurs de rein exprimaient DKK1. Les observations effectuées dans le cancer du poumon ont été, par la suite, corroborées par d'autres groupes qui ont montré une corrélation entre l'expression de DKK1 et un mauvais pronostic. Après avoir confirmée l’expression de DKK1 dans les cancers communs, justifiant ainsi sa candidature comme TAA, nous avons évalué l’immunogénicité de DKK1. Pour ce faire, nous avons effectué des stimulations in vitro de cellules mononucléées du sang périphérique (PBMC) de patient(e)s atteint(e)s d’un cancer du sein ou du poumon avec des peptides dérivés de DKK1 pouvant être présentés par les complexes majeurs d’histocompatibilité (CMH) HLA-A*0201. Des clones de T CD8+ reconnaissant un peptide de DKK1 ont été identifiés et isolés. Par essai multiplex et cytométrie de flux intracellulaire, la polyfonctionnalité d’un ces clones T CD8+ spécifiques à DKK1 a été étudiée et a révélée un profil effecteur, renforçant ainsi la candidature de DKK1 comme TAA. Dans l’ensemble, les résultats obtenus dans cette première partie de thèse suggèrent une possible utilisation de DKK1 en immunothérapie contre les cancers communs, attribuable à son expression dans ces cancers et la possibilité de faire proliférer des T CD8+ effecteurs spécifiques à DKK1 à partir de sang de patients. Dans la seconde partie de cette thèse, je décrirai la manipulation in vitro des T CD8+ de patients atteints d’un cancer commun, afin d’augmenter la force et la durée de leurs fonctions anti-tumorales. Il a été démontré que des lymphocytes moins différentiés sont capables d’une réponse immunologique plus efficace et durable. Nous avons basé ce projet sur l’utilisation d’un inhibiteur pharmacologique de la GSK-3, pour activer de la voie de Wnt chez les T CD8+ et ainsi leur conférer un phénotype moins différentié, partageant des caractéristiques de la cellule naïve et de la cellule mémoire. Des cultures de T CD8+, spécifiques à des antigènes viraux, en présence de l’inhibiteur ont permis d’augmenter la sécrétion d’interféron (IFN)- et leur activité cytotoxique. Ces résultats indiquent un effet de l’activation de la voie de Wnt sur la fonction des T CD8+. Ces observations sont rapportées pour la première fois chez les T CD8+ humains et suggèrent une nouvelle stratégie, applicables à l’immunothérapie du cancer, afin de prolonger la persistance des cellules ainsi que leur activité anti-tumorale. En conclusion, ces travaux de recherche ont mené à la réalisation d’une étape très importante dans la validation de la candidature de DKK1 comme TAA pour les cancers communs, soit la démonstration de son expression dans ces cancers et son absence dans les tissus normaux dérivés d’organes importants. Ces travaux ont également mené à la démonstration de l’immunogénicité de DKK1, par l’identification d’un peptide de DKK1 reconnu par les T CD8+. De plus, l’étude de la polyfonctionnalité des T CD8+ spécifiques à DKK1 a révélée un profil effecteur favorable pour l’obtention d’une réponse anti-tumorale efficace. Ces découvertes pourraient servir à l’élaboration d’une stratégie d’immunothérapie à médiation cellulaire pour les cancers communs. Pour sa part, l’étude phénotypique et fonctionnelle de la modulation de la voie de Wnt dans les T CD8+ a donné lieu à l’observation d’un phénotype encore jamais rapporté chez l’humain, conférant aux T CD8+ un aspect moins différentié avec des caractéristiques propre à un phénotype mémoire. Ces résultats sont pertinents dans l’amélioration de l’immunothérapie du cancer, passant par l’augmentation de la persistance des lymphocytes. En résumé, les résultats présentés dans cette thèse de doctorat fournissent des évidences indéniables quant à la validation de DKK1 comme TAA pour une immunothérapie à médiation cellulaire des cancers communs. Ces résultats fournissent également des preuves quant à la pertinence de la reprogrammation des T CD8+ par l’activation de la voie de la voie de Wnt, afin de générer des lymphocytes médiateurs plus efficaces pour ce type de thérapie.Cell-mediated cancer immunotherapy is based on the priming of the patient’s CD8+ T lymphocytes (CD8+ T cells) to mediate an immune response directed against the tumour. This anti-tumour response is antigen-specific and directed against tumour associated antigens (TAA), which are defined as proteins expressed principally by cancer cells and absent from non-malignant tissues. By utilizing a bio-informatic approach, we identified the gene DKK1, a Wnt pathway inhibitor, as a potential TAA. This was an important novel finding as the identification of a new TAA is one of the key elements to enhance cell-mediated cancer immunotherapy. Furthermore, patient treatment options may also be improved through the amplification of the force and duration of the anti-tumour immune response mediated by TAA specific T cells. This thesis is divided in two parts: 1- The characterization of DKK1 expression and immunogenicity in common cancers as validation of TAA candidate. 2- The reprogrammation of CD8+ T cells from patient with common cancers to restore a less-differentiated phenotype in an attempt to improve their anti-tumour response. We first characterized DKK1 expression in breast cancer and other common cancers. In order to prove its specificity to malignant tissues, the DKK1 expression profile was initially established by RT-PCR and ELISA assay using cancer cell lines and in RNA panels from normal tissues. DKK1 expression was also described using clinical samples from breast, lung and kidney cancers. We found that 30% of breast cancer clinical samples were positive for DKK1 expression. Interestingly, half of the triple negative breast cancer tumours (negative for the expression of progesterone and estrogen receptors and Her-2/neu) were DKK1 (+). Moreover, 50% of the lung cancer and 30% of the kidney cancer clinical samples were also DKK1 (+). These results have been corroborated by other groups who recently reported similar observations in lung cancer with a correlation with poor prognosis. After confirming that the DKK1 gene expression profile in common cancer qualifies DKK1 as a relevant TAA, we then explored its immunogenicity. To do so, we performed in vitro stimulations of peripheral blood mononuclear cells (PBMC) from lung and breast cancer patients with DKK1-derived synthetic peptides, which were selected for their capacity to be presented by the major histocompatibility complex (MHC) HLA-A*0201. With this method, we identified and isolated CD8+ T cell clones with a specificity unique for one DKK1 peptide. Cytokine secretion profile of anti-DKK1 T cells was established by cytokine mutiplex assay and flow cytometry. This study revealed that DKK1-specitfic CD8+ T cells had an effector profile with polyfunctionality proprieties, thereby reinforcing DKK1 as a TAA candidate. Altogether, the results obtained in this first part of this thesis suggest the possible use of DKK1 in common cancer immunotherapy as it is principally expressed by malignant tissues and can generate the activation of effector CD8+ T cells. In the second part of this thesis, I will describe in vitro manipulations of patients’ CD8+ T cells in the goal of augmenting their longevity and the strength of the anti-tumour response. Previous research revealed that a less-differentiated phenotype correlated with an augmented capacity of persistence and the intensity of the T cell response. For this project, we generated less-differentiated CD8+ T cells by activating the Wnt pathway with a pharmalogical inhibitor of GSK-3. These less-differentiated T cells shared a phenotype of both naive and memory T cells. As for their immune functions, viral antigen specific CD8+ T cells cultured with the inhibtitor showed an elevation in interferon (IFN)-γ production and cytotoxic activity. This represent the first report of such observations in humans CD8+ T cells and suggest a new stategy to prolonge the persistence of T cells in a cancer immunotherapy setting. In conclusion, this work has strongly contributed to the validation of DKK1 as a TAA for common cancers, as it is expressed in malignant tissues and relatively absent in form normal tissues. It demonstrated the immunogenicity of DKK1 with the identification of a DKK1 peptide recognized by CD8+ T cells. Moreover, these DKK1-specific CD8+ T cells appear to be polyfunctional with an effector profil, which is favorable to mount a potent anti-tumour response. These findings could serve in novel strategies to be exploited in cell-mediated immunotherapy against common cancers. Furthermore, the phenotypic and functional study of the Wnt pathway activation resulting in a less-differentiated CD8+ T cells, generated observations that had never been reported in humans. These findings are relevant for cancer immunotherapy because they could help generate less-differentiated cells with augmented persistance and anti-tumorale capacities. Altogether, the results presented in this doctoral thesis provide significant evidence that DKK1 may serve as a TAA in cell-mediated immunotherapy for patients affected by common cancers and that reprogramming of CD8+ T cells through activation of the Wnt pathway could generate more effective mediator for this type of treatment

Exploiting the neoantigen landscape for immunotherapy of pancreatic ductal adenocarcinoma

Immunotherapy approaches for pancreatic ductal adenocarcinoma (PDAC) have met with limited success. It has been postulated that a low mutation load may lead to a paucity of T cells within the tumor microenvironment (TME). However, it is also possible that while neoantigens are present, an effective immune response cannot be generated due to an immune suppressive TME. To discern whether targetable neoantigens exist in PDAC, we performed a comprehensive study using genomic profiles of 221 PDAC cases extracted from public databases. Our findings reveal that: (a) nearly all PDAC samples harbor potentially targetable neoantigens; (b) T cells are present but generally show a reduced activation signature; and (c) markers of efficient antigen presentation are associated with a reduced signature of markers characterizing cytotoxic T cells. These findings suggest that despite the presence of tumor specific neoepitopes, T cell activation is actively suppressed in PDAC. Further, we identify iNOS as a potential mediator of immune suppression that might be actionable using pharmacological avenues

Cervical Cancer Neoantigen Landscape and Immune Activity is Associated with Human Papillomavirus Master Regulators

Human papillomaviruses (HPVs) play a major role in development of cervical cancer, and HPV oncoproteins are being targeted by immunotherapies. Although these treatments show promising results in the clinic, many patients do not benefit or the durability is limited. In addition to HPV antigens, neoantigens derived from somatic mutations may also generate an effective immune response and represent an additional and distinct immunotherapy strategy against this and other HPV-associated cancers. To explore the landscape of neoantigens in cervix cancer, we predicted all possible mutated neopeptides in two large sequencing data sets and analyzed whether mutation and neoantigen load correlate with antigen presentation, infiltrating immune cell types, and a HPV-induced master regulator gene expression signature. We found that targetable neoantigens are detected in most tumors, and there are recurrent mutated peptides from known oncogenic driver genes (KRAS, MAPK1, PIK3CA, ERBB2, and ERBB3) that are predicted to be potentially immunogenic. Our studies show that HPV-induced master regulators are not only associated with HPV load but may also play crucial roles in relation to mutation and neoantigen load, and also the immune microenvironment of the tumor. A subset of these HPV-induced master regulators positively correlated with expression of immune-suppressor molecules such as PD-L1, TGFB1, and IL-10 suggesting that they may be involved in abrogating antitumor response induced by the presence of mutations and neoantigens. Based on these results, we predict that HPV master regulators identified in our study might be potentially effective targets in cervical cancer

Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues

Abstract Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision

Stimulation of Wnt/ß-Catenin Pathway in Human CD8⁺ T Lymphocytes from Blood and Lung Tumors Leads to a Shared Young/Memory Phenotype

<div><p>Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8⁺ T cells towards stem cell-like memory (T_SCM) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T_SCM can be obtained from human mature CD8⁺ T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8⁺ T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L⁺CD45RA⁺ cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T_SCM cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T_SCM CD8⁺ T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T_SCM subset in human CD8⁺ T cells from TIL and the periphery, which are relevant for ACT.</p> </div

A minimal concentration of 2.5 µM of TWS119 is required to generate T cells with a young/memory phenotype.

<p>Expression of CD45RA and CD62L on CD8⁺ T cells from peripheral blood of healthy donors after activation of the Wnt pathway with TWS119 dose-response (0.5 to 5 µM). (<b>A</b>) Expansion of viable CD8⁺ T cells activated with anti-CD3/IL-2 after five days of culture with or without 5 µM TWS119, illustrated by final cell concentration. Initial cell concentration was 1×10⁶ cell/mL. (<b>B</b>) Representative dot plot analysis for surface expression of CD45RA and CD62L on CD8⁺ T cells activated with anti-CD3/IL-2 with or without increasing concentrations of TWS119. (<b>C</b>) Percentage of total CD8⁺ T cells in each CD45RA/CD62L population for 6 healthy donors. (<b>D</b>) Percentage of CD8⁺ T cells expressing CD127 in total CD8⁺ T cell population in a TWS119 dose-response, in each population defined by expression of CD45RA and CD62L in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a> (for 6 donors).</p

Activation of human CD8⁺ T cells in the presence of TWS119 favors a young/memory phenotype.

<p>CD127 and CD133 expression in CD8⁺ T cell sub-populations defined by expression of CD45RA and CD62L (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a>) following treatment with TWS119. (<b>A</b>) Representative histograms of total CD8⁺ T cells expressing CD127 or CD133 after a 5-day anti-CD3/IL-2 activation with or without TWS119. (<b>B–C</b>) Percentage of CD8⁺ T cells expressing, respectively, CD127 (<b>B</b>) and CD133 (<b>C</b>) in total CD8⁺ T cell population (Total), and in each CD45RA/CD62L-defined population from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a>, for 9 healthy donors in B and 5 in C. (<b>D</b>) Percentage of CD8⁺ T cells expressing both CD127 and CD133 markers in total CD8⁺ T cell population (total) and in each CD45RA/CD62L-defined population from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a> for 5 healthy donors. * <i>P</i><0.004.</p