Evaluation of an antiviral peptide immunotherapy strategy in nasopharyngeal carcinoma associated with Epstein Barr virus : From in vitro experimentation up to proposal of new therapeutic trials

Le carcinome du nasopharynx (CNP) est associé à la latence II d’EBV et serait une cible préférentielle de peptides vaccinaux, dérivés des antigènes de latence II d’EBV, au sein d’une approche d’immunothérapie ciblant une réponse de type Th1. Cependant, les cellules de CNP sécrétaient des exosomes immunosuppresseurs qui induisent la mort des cellules T CD4+ Th1. En outre, cet environnement immunosuppressif est aggravé par un recrutement important de lymphocytes T régulateurs (Treg) qui sont associés à un mauvais pronostic dans le CNP. Ainsi, les objectifs de ma thèse ont consisté à (i) étudier l’impact des exosomes tumoraux de CNP (Exo-CNP) sur le phénotype et la fonctionnalité des Treg (ii) explorer les moyens de favoriser l’expansion et l’efficacité des effecteurs anti-tumoraux, notamment des lymphocytes T CD4+ Th1 anti-EBV, en proposant une stratégie d’immunothérapie peptidique. Dans la première partie, nous avons montré in vivo que la chimiokine CCL-20 était responsable du recrutement des Treg au sein de la tumeur. Par ailleurs, nous avons montré que les exosomes de CNP expriment la CCL-20 et sont capables de favoriser in vitro l’attraction des Treg via cette chimiokine. En parallèle, nous avons montré que les Exo-CNP (i) augmentent significativement l’expansion des Treg, (ii) augmentent significativement le niveau d’expression du CD25 et du FoxP3 sur les Treg, (iv) conduisent à la conversion des cellules T CD4+CD25neg en Treg CD4+CD25high. L’ensemble de ces résultats a été corrélé à une augmentation significative de l’activité suppressive des Treg en présence des exosomes de CNP. Dans la deuxième partie, nous avons validé ex vivo l’immunogénicité des peptides sur des cellules immunitaires humaines. Les résultats obtenus montrent également que des lignées T CD4+ humaines spécifiques d’EBV induisent une lyse des cellules de CNP EBV+. Et, de façon inattendue, la présence des Exo-CNP ne semble pas altérer les capacités de lyse des lignées. Enfin, nous avons pu valider l’efficacité des peptides dans un modèle in vivo. En effet, nous avons évalué l’effet anti-tumoral de la vaccination par les peptides dérivés d’EBV en mesurant l’évolution des masses tumorales. Nous avons pu montrer que la reconstitution immunitaire des souris SCID-CNP couplée à la vaccination peptidique permet la stabilisation de la croissance tumorale au cours du temps. En conclusion, la mise en évidence d’une coopération entre les Exo-CNP et les Treg est très importante et pourrait représenter ainsi un nouveau mécanisme d’échappement du CNP au système immunitaire. Par ailleurs, nos résultats consolident l’efficacité des peptides EBV qui pourraient être utilisés, à terme, dans une stratégie de vaccination peptidique ou de thérapie cellulaire en complément des traitements de référence chez les patients atteints d’un CNP.Nasopharyngeal carcinoma (NPC) is associated to the EBV latency II and should be a preferential target for our previously selected peptides derived from EBV latency II antigens. However, previous studies performed showed that NPC cells release exosomes which are able to induce apoptosis in mature Th1 lymphocytes. Furthermore, the NPC immunosuppressive environment is compounded by significant recruitment of natural regulatory T cells (Treg) associated with a poor prognosis in NPC. Thereby, this immunosuppressive environment could potentially be an obstacle to the use of this promiscious peptide coktail as a future vaccine in EBV associated nasopharyngeal carcinoma. In this context, the main objectives of my thesis consisted of (i) investigating the impact of NPc-derived-exosomes and Treg and (ii) exploring ways to promote expansion and efficiency of anti-tumoral effectors, notably anti-EBV Th1 CD4+ lymphocytes. First, we showed for the first time that NPC exosomes contain CCL-20 chemokine and are able to facilitate the recruitment of Treg cells in a CCL-20 dependent manner. This finding was confirmed by in vivo experiments in immunodeficient (SCID) and humanized mouse model xenografted with human NPC. NPC exosomes significantly increase the expansion, significantly increase the expression level of CD25 and FoxP3 on Treg and lead to the conversion of CD4+CD25neg T cells into CD4+CD25high Treg. These converted cells showed an effective immunosuppressive activity on autologous PBMCs. Moreover, NPC exosomes induce Treg suppressive activity and recruitment. In the second part, we validated peptides immunogenicity ex vivo. Then, we showed efficient EBV specific T CD4+ cytotoxicity in NPC cells. Furthermore, tumor exosomes do not affect the lysis capacity of our generated EBV specific T CD4+ cell lines. Finally, the efficiency of the EBV peptides coktail was evaluated in vivo by injecting EBV peptides into SCID mice xenografted with human NPC cells and reconstituted with human PBMCs. Here we showed that immune reconstitution coupled to peptide vaccination could stabilize tumor growth. In conclusion, our findings give new insights about NPC-derived exosomesimmunoregulatory properties and we showed that interactions of NPC-exosomes with Treg cells support immune evasion of human NPC. Our results also confirm that EBV peptides coktail could eventually be used in a vaccination strategy in combination with standard treatments in NPC patients to minimize relapse and control residual disease

Évaluation d'une stratégie d'immunothérapie peptidique antivirale dans le carcinome du nasopharynx associé au virus d'Epstein Barr (de l'expérimentation in vitro à la proposition de nouveaux essais thérapeutiques)

Le carcinome du nasopharynx (CNP) est associé à la latence II d EBV et serait une cible préférentielle de peptides vaccinaux, dérivés des antigènes de latence II d EBV, au sein d une approche d immunothérapie ciblant une réponse de type Th1. Cependant, les cellules de CNP sécrétaient des exosomes immunosuppresseurs qui induisent la mort des cellules T CD4+ Th1. En outre, cet environnement immunosuppressif est aggravé par un recrutement important de lymphocytes T régulateurs (Treg) qui sont associés à un mauvais pronostic dans le CNP. Ainsi, les objectifs de ma thèse ont consisté à (i) étudier l impact des exosomes tumoraux de CNP (Exo-CNP) sur le phénotype et la fonctionnalité des Treg (ii) explorer les moyens de favoriser l expansion et l efficacité des effecteurs anti-tumoraux, notamment des lymphocytes T CD4+ Th1 anti-EBV, en proposant une stratégie d immunothérapie peptidique. Dans la première partie, nous avons montré in vivo que la chimiokine CCL-20 était responsable du recrutement des Treg au sein de la tumeur. Par ailleurs, nous avons montré que les exosomes de CNP expriment la CCL-20 et sont capables de favoriser in vitro l attraction des Treg via cette chimiokine. En parallèle, nous avons montré que les Exo-CNP (i) augmentent significativement l expansion des Treg, (ii) augmentent significativement le niveau d expression du CD25 et du FoxP3 sur les Treg, (iv) conduisent à la conversion des cellules T CD4+CD25neg en Treg CD4+CD25high. L ensemble de ces résultats a été corrélé à une augmentation significative de l activité suppressive des Treg en présence des exosomes de CNP. Dans la deuxième partie, nous avons validé ex vivo l immunogénicité des peptides sur des cellules immunitaires humaines. Les résultats obtenus montrent également que des lignées T CD4+ humaines spécifiques d EBV induisent une lyse des cellules de CNP EBV+. Et, de façon inattendue, la présence des Exo-CNP ne semble pas altérer les capacités de lyse des lignées. Enfin, nous avons pu valider l efficacité des peptides dans un modèle in vivo. En effet, nous avons évalué l effet anti-tumoral de la vaccination par les peptides dérivés d EBV en mesurant l évolution des masses tumorales. Nous avons pu montrer que la reconstitution immunitaire des souris SCID-CNP couplée à la vaccination peptidique permet la stabilisation de la croissance tumorale au cours du temps. En conclusion, la mise en évidence d une coopération entre les Exo-CNP et les Treg est très importante et pourrait représenter ainsi un nouveau mécanisme d échappement du CNP au système immunitaire. Par ailleurs, nos résultats consolident l efficacité des peptides EBV qui pourraient être utilisés, à terme, dans une stratégie de vaccination peptidique ou de thérapie cellulaire en complément des traitements de référence chez les patients atteints d un CNP.Nasopharyngeal carcinoma (NPC) is associated to the EBV latency II and should be a preferential target for our previously selected peptides derived from EBV latency II antigens. However, previous studies performed showed that NPC cells release exosomes which are able to induce apoptosis in mature Th1 lymphocytes. Furthermore, the NPC immunosuppressive environment is compounded by significant recruitment of natural regulatory T cells (Treg) associated with a poor prognosis in NPC. Thereby, this immunosuppressive environment could potentially be an obstacle to the use of this promiscious peptide coktail as a future vaccine in EBV associated nasopharyngeal carcinoma. In this context, the main objectives of my thesis consisted of (i) investigating the impact of NPc-derived-exosomes and Treg and (ii) exploring ways to promote expansion and efficiency of anti-tumoral effectors, notably anti-EBV Th1 CD4+ lymphocytes. First, we showed for the first time that NPC exosomes contain CCL-20 chemokine and are able to facilitate the recruitment of Treg cells in a CCL-20 dependent manner. This finding was confirmed by in vivo experiments in immunodeficient (SCID) and humanized mouse model xenografted with human NPC. NPC exosomes significantly increase the expansion, significantly increase the expression level of CD25 and FoxP3 on Treg and lead to the conversion of CD4+CD25neg T cells into CD4+CD25high Treg. These converted cells showed an effective immunosuppressive activity on autologous PBMCs. Moreover, NPC exosomes induce Treg suppressive activity and recruitment. In the second part, we validated peptides immunogenicity ex vivo. Then, we showed efficient EBV specific T CD4+ cytotoxicity in NPC cells. Furthermore, tumor exosomes do not affect the lysis capacity of our generated EBV specific T CD4+ cell lines. Finally, the efficiency of the EBV peptides coktail was evaluated in vivo by injecting EBV peptides into SCID mice xenografted with human NPC cells and reconstituted with human PBMCs. Here we showed that immune reconstitution coupled to peptide vaccination could stabilize tumor growth. In conclusion, our findings give new insights about NPC-derived exosomesimmunoregulatory properties and we showed that interactions of NPC-exosomes with Treg cells support immune evasion of human NPC. Our results also confirm that EBV peptides coktail could eventually be used in a vaccination strategy in combination with standard treatments in NPC patients to minimize relapse and control residual disease.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Control of the Inflammatory Response Mechanisms Mediated by Natural and Induced Regulatory T-Cells in HCV-, HTLV-1-, and EBV-Associated Cancers

Virus infections are involved in chronic inflammation and, in some cases, cancer development. Although a viral infection activates the immune system’s response that eradicates the pathogen mainly through inflammatory mechanisms, it is now recognized that this inflammatory condition is also favorable to the development of tumors. Indeed, it is well described that viruses, such as hepatitis C virus (HCV), Epstein Barr virus (EBV), human papillomavirus (HPV) or human T-cell lymphotropic virus type-1 (HTLV-1), are important risk factors for tumor malignancies. The inflammatory response is a fundamental immune mechanism which involves several molecular and cellular components consisting of cytokines and chemokines that are released by various proinflammatory cells. In parallel to this process, some endogenous recruited components release anti-inflammatory mediators to restore homeostasis. The development of tools and strategies using viruses to hijack the immune response is mostly linked to the presence of regulatory T-cells (Treg) that can inhibit inflammation and antiviral responses of other effector cells. In this review, we will focus on current understanding of the role of natural and induced Treg in the control and the resolution of inflammatory response in HCV-, HTLV-1-, and EBV-associated cancers

Activation of a Helper and Not Regulatory Human CD4+ T Cell Response by Oncolytic H-1 Parvovirus

International audienceBackground: H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment. Methodology/Principal findings: Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-c and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. Conclusions/Significance: Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment

Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells

Comment in :- RE: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells. [J Natl Cancer Inst. 2015]- Response. [J Natl Cancer Inst. 2015]International audienceBACKGROUND: Regulatory T cells (Treg) and tumor-exosomes are thought to play a role in preventing the rejection of malignant cells in patients bearing nasopharyngeal carcinoma (NPC).METHODS: Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients' plasma (Patient-Exo), and CCL20 were tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model (n = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA, and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). The Student's t test was used for group comparisons. All statistical tests were two-sided.RESULTS: CCL20 allowed the intratumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34 fold increase, P < .001), which was statistically significantly inhibited (P < .001) by an anti-CCL20 blocking mAb. NPC-Exo also recruited conventional CD4(+)CD25(-) T cells and mediated their conversion into inhibitory CD4(+)CD25(high) cells. Moreover, NPC-Exo enhanced (P = .0048) the expansion of human Treg, inducing the generation of Tim3(Low) Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75 (P < .001). These results were consistent with a stronger suppression of responder cells' proliferation and the secretion of immunosuppressive cytokines (IL10, TGFB1).CONCLUSION: Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC

H-1 PV exerts its effect on PHA-activated PBMCs in an NS1-dependent manner.

<p>PBMCs were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. <b>A</b>. <i>H-1 PV alters PHA-activated PBMC morphology</i>. For each condition, pictures of cells were taken in the course of infection but only those taken 48 h post-infection are shown. Scale bar = 100 µm. <b>B</b>. <i>PHA-activated PBMCs are lysed upon H-1 PV infection</i>. H-1 PV cytotoxicity was assessed using a test based on a bioluminescent reaction measuring the leak of a cellular marker from cytoplasm to culture medium, which reveals the loss of plasma membrane integrity. Assays were performed at different times after H-1 PV inoculation but only 48 and 72 h conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). <b>C</b>. <i>PHA-activated PBMC death upon H-1 PV infection is related to NS1 protein expression but does not depend on caspase 3 activation</i>. Non activated (NA) and activated (A) PBMC total protein extracts were prepared in the course of infection. Equal amounts of proteins were separated by 4–12% SDS-PAGE and analyzed by Western blot for the presence of NS1 protein, cleaved caspase 3 and both full-length and cleaved PARP. β-actin was used as a loading control. <b>D</b>. <i>PBMCs do not support an H-1 PV productive infection</i>. Both PBMCs and culture supernatants were collected and tested for the presence of infectious viral particles using the Tissue Culture Infectious Dose 50 method. The graph represents the time course of both released and non-released virions amounts which were added and expressed in plaque forming unit (pfu). <i>Representative data from 3 independent experiments</i>.</p

H-1 PV affects neither Treg cell phenotype nor viability while inhibiting their suppressive activity.

<p>Treg cells were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. <b>A</b>. <i>Control of Treg cell purity</i>. Cells were labelled for Treg cell markers and analyzed using flow cytometry. Results are expressed as percentages of CD4+CD25+, CD4+CD25+CD127- and CD4+CD25+FoxP3+ cells within the whole cell population. <b>B</b>. <i>Treg cells do not undergo any major phenotypical changes upon infection</i>. Cells were labelled for Treg cell markers and analyzed using flow cytometry 48 h p.i. Results are expressed as percentages of CD4+CD25+ and CD4+CD25+CD127- cells within the whole cell population. <b>C</b>. <i>H-1 PV does not affect Treg cell viability</i>. Cell viability was assessed 24 and 48 h p.i using a bioluminescent test measuring ATP amount in living cells. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). <b>D</b>. <i>Ex vivo Treg cells are able to suppress autologous activated PBMCs proliferation</i>. Autologous PBMCs were activated (anti-CD3 and -CD28 antibodies) and cultured with increasing quantities of Treg cells. PBMCs proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA 48 h p.i. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). The black arrow indicates the ratio chosen for further experiments. <b>E</b>. <i>H-1 PV is able to inhibit Treg cell suppressive activity</i>. Autologous PBMCs were activated (anti-CD3 and -CD28 antibodies), cultured with Treg cells (4 PBMCs for 2 Treg cells) and mock- or H-1PV-treated. PBMC proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA 48 h p.i. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). <i>Representative data from 3 independent experiments</i>.</p