Privatization, cooperation and costs of solid waste services in small towns

This paper analyzes the cost implications of privatization and cooperation in the provision of solid waste services for a sample of small municipalities. In conducting this empirical analysis, a survey is first designed and administered to municipalities in the Spanish region of Aragon, and then an estimation of the determinants of service costs is undertaken, considering the possible endogeneity of delivery choices. Our findings indicate that cooperation is more effective than privatization in saving costs. Both production forms can enable small municipalities to cut costs by exploiting scale economies. However, the fact that inter-municipal cooperation involves lower transaction costs and is less likely to be affected by competition problems would seem to account for the fact that it is a more effective way of reducing costs.

Spleen Volume Variation in Patients with Locally Advanced Non-Small Cell Lung Cancer Receiving Platinum-Based Chemo-Radiotherapy.

There is renewed interest in the immune regulatory role of the spleen in oncology. To date, very few studies have examined macroscopic variations of splenic volume in the setting of cancer, prior to or during therapy, especially in humans. Changes in splenic volume may be associated with changes in splenic function. The purpose of this study was to investigate variations in spleen volume in NSCLC patients during chemo-radiotherapy. Sixty patients with stage I-IIIB NSCLC underwent radiotherapy (60Gy/30 fractions) for six weeks with concomitant carboplatin/paclitaxel (Ca/P; n = 32) or cisplatin/etoposide (Ci/E; n = 28). A baseline PET/CT scan was performed within 2 weeks prior to treatment and during Weeks 2 and 4 of chemo-radiotherapy. Spleen volume was measured by contouring all CT slices. Significant macroscopic changes in splenic volume occurred early after the commencement of treatment. A significant decrease in spleen volume was observed for 66% of Ca/P and 79% of Ci/E patients between baseline and Week 2. Spleen volume was decreased by 14.2% for Ca/P (p<0.001) and 19.3% for Ci/E (p<0.001) patients. By Week 4, spleen volume was still significantly decreased for Ca/P patients compared to baseline, while for Ci/E patients, spleen volume returned to above baseline levels. This is the first report demonstrating macroscopic changes in the spleen in NSCLC patients undergoing radical chemo-radiotherapy that can be visualized by non-invasive imaging.

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis.

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.

Cyclooxygenase-2 Expression in Non-Small Cell Lung Cancer Correlates With Hypertrophic Osteoarthropathy.

Hypertrophic osteoarthrpathy (HO) is a rare paraneoplasic syndrome associated with non-small cell lung cancer (NSCLC). The pathophysiology of HO is unknown but was recently related to enhanced levels of urine prostaglandin E2 (PGE2). Here, we report the case of a patient that presented HO in association with a resectable left upper lobe NSCLC. Following surgery and adjuvant chemotherapy, HO resolved and did not recur with development of a brain metastasis 1 year later. Interestingly, tumor cyclooxygenase-2, an enzyme responsible the synthesis of PGE2, was expressed in the primary tumor but not in the resected metastasis.

Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens.

Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR-pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR-pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering.

Corporate Social and Regional Responsibility - The View of Small and Medium-sized Enterprises

The objective of this research was to understand and describe what corpo-rate social and regional responsibility is in SMEs and define the meaning of these concepts to the community and region. Corporate social respon-sibility (CSR) creates a basis for regional responsibility. Regional respon-sibility is a new concept and this research examines it from SMEs’ view-point. This is a theoretical research and the aim is to create a theoretical framework of SMEs’ corporate social and regional responsibility. This framework supports the future research on the subject. The research results show that CSR of SMEs is practical, informal and dependent on the scarce resources of SMEs. CSR is a complex and deep concept and SMEs have their own way of interpreting it. It can be stated that CSR-practises in SMEs are closely connected to employment, envi-ronment, community and supply chain. The challenge is to find motivation to socially and regionally responsible behaviour in SMEs. Benefiting from responsible behaviour and the attitude of SME’s owner-manager are the key reasons for SMEs to involve in CSR and regional responsibility. The benefits of this involvement are for example improved image, reputation and market position. CSR can also be used in SMEs as risk management tool and in cost reduction. This study indicates also that creation of strate-gic partnerships, local government participation, a proper legal system and financial support are the basic issues which support CSR of SMEs. This research showed that regional responsibility of SMEs includes active participation in regional strategy processes, L&RED initiatives and regional philanthropy. For SMEs regional responsibility means good relationships with the community and other related stakeholders, involvement in L&RED initiatives and acting responsibly towards the operating environment. In SMEs’ case this means that they need to understand the benefits of this kind of involvement in order to take action and participate. As regional responsibility includes the relationships between firm and the community, it can be stated that regional responsibility extends CSR’s view of stakeholders and emphasises both, the regional stakeholders and public-private partnerships. Community engagement and responsible be-haviour towards community can be seen as a part of SMEs’ social and regional responsibility. This study indicates that social and regional re-sponsibility of SMEs have a significant influence on the community and region where they are located. Better local and regional relationships with regional and community actors are the positive impacts of social and re-gional responsibility of SMEs. Socially and regionally responsible behav-iour creates a more positive environment and deepens the involvement of SMEs to community and L&RED initiatives.

Prospective evaluation of circulating VEGF in patients with advanced non-small cell lung cancer treated with bevacizumab, pemetrexed and cisplatin in the trial SAKK19/09.

Aim: Bevacizumab is a monoclonal antibody directed against the vascular endothelial growth factor (VEGF). The previous phase II trial ABIGAIL (Reck, 2010) suggested circulating VEGF as a prognostic, but not predictive, biomarker for patients (pts) with non-small cell lung cancer (NSCLC) treated with bevacizumab. We prospectively measured VEGF in the multicenter phase II trial SAKK19/09 (NCT01116219). Methods: SAKK19/09 enrolled 77 evaluable patients (pts) with previously untreated, advanced nonsquamous NSCLC and EGFR wild type. Pts received 4 cycles of cisplatin 75mg/m2 (or carboplatin AUC5), pemetrexed 500mg/m2 and bevacizumab 7.5mg/kg, followed by maintenance therapy with pemetrexed and bevacizumab until progression by RECIST1.1. Follow-up CT scans were performed every 6 weeks until week 54 and every 12 weeks thereafter. Baseline EDTA blood samples were sent by same-day courier to the central laboratory for centrifugation, aliquoting, and freezing. Upon completion of enrollment, aliquots were thawed, and VEGF quantification was performed centrally using Luminex® Performance Assay Human Base Kit A (R&D Systems, Abingdon, UK). The mean value was used to stratify pts into two groups (low versus high VEGF). Best response rate assessed by RECIST1.1 (CR + PR versus SD + PD). Results: Clinical results of the SAKK19/09 trial were reported previously (Gautschi, 2013). Baseline plasma VEGF was detectable in 71 of 77 (92%) evaluable patients treated with chemotherapy and bevacizumab. The mean value was 74.9 pg/ml, the median 47.5 pg/ml, and the range 3.55 to 310 pg/ml. Using the mean as a predefined cutoff value, 50 patients had low VEGF levels and 21 patients had high VEGF levels. High VEGF was significantly associated with shorter PFS (4.1 vs 8.3 months, HR = 2.56; 95%CI: 1.43- 4.57; p = 0.0015) and OS (8.7 vs 17.5 months, HR = 2.67; 95% CI: 1.37-5.20; p = 0.0041), but not with best response rate ( p = 0.2256). Conclusions: Consistent with the ABIGAIL trial, circulating VEGF was prognostic, but not predictive for response, in the current trial. Further work is ongoing to identify potentially predictive biomarkers for bevacizumab, using comprehensive proteomic analyses. Disclosure: S.I. Rothschild: I received honoraria for the participation in advisory boards from Eli Lilly and Roche and for presentations at scientific symposiums sponsored by Roche; O. Gautschi: Honoraria for advisory boards of Eli Lilly and Roche; R. Cathomas: Advisory board member: Eli Lilly. All other authors have declared no conflicts of interest.

Prevalence and clinical outcomes for patients with MET protein expression in patients with non-small cell lung cancer in Europe: Results from the European Thoracic Oncology Platform Lungscape Project.

Aim The reported prevalence of MET overexpression varies from 25-55% in non-small cell lung cancer (NSCLC) and clinical correlations are emerging slowly. In a well-defined NSCLC cohort of the Lungscape program, we explore the epidemiology, the natural history of IHC MET positivity and its association to OS, RFS and TTR. Methods Resected stage I-III NSCLC identified based on the quality of clinical data and FFPE tissue availability were assessed for MET expression using immunohistochemistry (IHC) on TMAs (CONFIRM anti total c-MET assay, clone SP44, Ventana BenchMark platform). All cases were analysed at participating pathology laboratories using the same protocol, after passing an external quality assurance program. MET positive status is defined as ≥ 50% of tumor cells staining with 2+ or 3+ intensity. Results A total of 2709 cases are included in the iBiobank and will be analysed. IHC MET expression is currently available for 1552 patients, with positive MET IHC staining in 380 cases [24.5%; IHC 3+ in 157 cases (41.3%) and 2+ in 223 cases (58.7%)]. The cohort of 1552 patients includes 48.2%, 44.7% and 4.4% cases of adenocarcinoma, squamous and large cell histologies, respectively. IHC MET status was independent of stage, age and smoking history. Significant differences in MET positivity were associated with gender (32% vs. 21% for female vs. male, p < 0.001), with performance status (25% vs. 18% for 0 vs. 1-3, p = 0.006), and histology (34%, 14% and 24% for adenocarcinoma, squamous and large cell carcinoma, p < 0.001). IHC MET positivity was independent of the IHC ALK status (p = 0.08). At last FU, 52% of patients were still alive, with a median FU of 4.8 yrs. No association of IHC MET was found with OS, RFS or TTR. Conclusions The preliminary results for this large multicentre European cohort describe a prevalence of MET overexpression that seems lower than previous observations in NSCLC, such as reported for the OAM4971g trial, suggesting potential biological differences between surgically resected and metastatic disease. Analysis for the full cohort is ongoing and results will be presented. Disclosure L. Bubendorf: Disclosures: Stock ownership: Roche Advisory boards: Roche, Pfizer Research support: Roche; K. Schulze: Full time employee of Roche; A. Das-Gupta: I am a full time employee of Roche. All other authors have declared no conflicts of interest.

Final results of the SAKK 16/00 trial: a randomized phase III trial comparing neoadjuvant chemoradiation to chemotherapy alone in stage IIIA/N2 non-small cell lung cancer (NSCLC).

Aim: One standard option in the treatment of stage IIIA/N2 NSCLC is neoadjuvant chemotherapy followed by surgery. We investigated in a randomized trial whether the addition of neoadjuvant radiotherapy would improve the outcome. Here we present the final results of this study. Methods: Patients (pts.) with pathologically proven, resectable stage IIIA/N2 NSCLC, performance status 0-1, and adequate organ function were randomized 1:1 to chemoradiation (CRT) with 3 cycles of neoadjuvant chemotherapy (cisplatin 100 mg/m2 and docetaxel 85 mg/m2 d1, q3weeks) followed by accelerated concomitant boost radiotherapy (RT) with 44 Gy in 22 fractions in 3 weeks, or neoadjuvant chemotherapy alone (CT), with subsequent surgery for all pts. The primary endpoint was event-free survival (EFS). Results: 232 pts. were randomized in 23 centers, the median follow-up was 53 months. Two thirds were men, median age was 60 years (range 37-76). Histology was squamous cell in 33%, adenocarcinoma in 43%. Response rate to CRT was 61% vs. 44% with CT. 85% of all pts. underwent surgery, 30-day postoperative mortality was 1%. The rate of complete resection was 91% (CRT) vs. 81% (CT) and the pathological complete remission (pCR) rate was 16% vs. 12%. The median EFS was 13.1 months (95% CI 9.9 - 23.5) for the CRT group vs. 11.8 months (95% CI 8.4 - 15.2) in the CT arm (p 0.665). The median overall survival (OS) with CRT was 37.1 months (95% CI 22.6 -50), with CT 26.1 months ( 95% CI 26.1 - 52.1, p 0.938). The local failure rate was 23% in both arms. In the CT arm 12 pts. were given postoperative radiotherapy (PORT) for R1 resection, 6 pts. received PORT in violation of the protocol. Pts. with a pCR, mediastinal downstaging to ypN0/1 and complete resection had a better outcome. Toxicity of chemotherapy was substantial, especially febrile neutropenia was common, whereas RT was well tolerated. Conclusions: This is the first completed phase III trial to evaluate the role of induction chemoradiotherapy and surgery, in comparison to neoadjuvant CT alone followed by surgery. RT was active, it increased response, complete resection and pCR rates. However, this failed to translate into an improvement of local control, EFS or OS. Notably, surgery after induction treatment was safe, including pneumonectomy. The overall survival rates of our neoadjuvant regimen are very encouraging, especially for a multicenter setting. Disclosure: M. Pless: Advisory Board for Sanofi; R. Cathomas: Advisory Board Sanofi D.C. Betticher: Advisory Board Sanofi. All other authors have declared no conflicts of interest.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Further molecular profiling of tumors harboring therapeutic targets within non-small cell lung cancer

Background: Treatment of NSCLC has been revolutionized in recent years with the introduction of several targeted therapies for selected genetically altered subtypes of NSCLC. A better understanding of molecular characteristics of NSCLC, which features common drug targets, may identify new therapeutic options. Methods: Over 6,700 non-small cell lung cancer cases referred to Caris Life Sciences between 2009 and 2014. Diagnoses and history were collected from referring physicians. Specific testing was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification/rearrangement (CISH or FISH), and/or RNA fragment analysis. Results: Tumors profiles from patients with hormone receptor positive disease (HER2, ER, PR, or AR positive by IHC) (n=629), HER2 mutations (n=8) ALK rearrangements (n=55), ROS1 rearrangement (n=17), cMET amplification or mutation (n=126), and cKIT mutation (n=11) were included in this analysis and compared to the whole cohort. Tumors with ALK rearrangement overexpressed AR in 18% of cases, and 7% presented with concomitant KRAS mutation. Lower rates of PTEN loss, as assessed by IHC, were observed in ALK positive (20%), ROS1 positive (9%) and cKIT mutated tumors (25%) compared to the overall NSCLC population (58%). cMET was overexpressed in 66% of ROS1 translocated and 57% of HER2 mutated tumors. cKIT mutations were found co-existing with APC (20%) and EGFR (20%) mutations. Pathway analysis revealed that hormone receptor positive disease carried more mutations in the ERK pathway (32%) compared to 9% in the mTOR pathway. 25% of patients with HER2 mutations harbored a co-existing mutation in the mTOR pathway. Conclusions: Pathway profiling reveals that NSCLC tumors present more often than reported with several concomitant alterations affecting the ERK or AKT pathway. Additionally, they are also characterized by the expression of potential biological modifiers of the cell cycle like hormonal receptors, representing a rationale for dual inhibition strategies in selected patients. Further refining of the understanding of NSCLC biomarker profile will optimize research for new treatment strategies.

Molecular profiling of non-small cell lung cancer by histologic subtype

Background: A substantial proportion of NSCLC has been shown to harbour specific molecular alterations affecting tumour proliferation and resulting in sensitivity to inhibition of the corresponding activated oncogenic pathway by targeted therapies. Comprehensive tumor profiling can diagnose such alterations and may identify new alterations opening additional treatment options for all distinct NSCLC subtypes. Methods: Over 6,700 non-small cell lung cancer cases referred to Caris Life Sciences between 2009 and 2014 were evaluated; clinical diagnoses and detailed tumor pathology were collected from referring physicians. Specific profiling was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification/rearrangement (CISH or FISH), and/or RNA fragment analysis within potential cancer-related genes and pathways. Results: Patients were grouped into cohorts according to histological subtype - adenocarcinoma (AD) (n=4,286), squamous cell carcinoma (SCC) (n=1,280), large cell carcinoma (LCC) (n=153) and bronchioalveolar carcinoma (BAC) (n=94). Protein overexpression of cMET (>2+ in >50% cells) was higher in AD (35.9%) compared to other subgroups (12-20%) while RRM1 and TOP2A levels were lower in AD. ALK or ROS1 were rearranged in 5.3% of patients with AD compared to 3.7% of patients with LCC and 1.2% of patients with SCC. EGFR mutations were found at low prevalence in both the LCC (0%) and SCC cohorts (2.8%) compared to 21% in AD. Similar lower rates of BRAF mutations were observed in the LCC and SCC cohorts compared to AD (0%, 1.1% and 5.1%). Pathway analysis showed activating mutations in the ERK pathway in 40% of patients with AD. Only 10-12% of patients with LCC or SCC had activating mutations in the ERK pathway. Conclusions: Despite the limitations of this retrospective series, we report comprehensive profiling of the largest cohort of NSCLC. Tumor profiling reveals that ADs may be more addicted to the ERK pathway than other histological subtypes. Drugs which target cMET may also have most utility in AD. Full analysis by histological subtype and additional correlative data on protein expression, gene copy number and mutations will be presented.

The role of renal biopsy in small renal masses.

Renal biopsy is being increasingly proposed as a diagnostic tool to characterize small renal masses (SRM). Indeed, the wide adoption of imaging in the diagnostic workup of many diseases had led to a substantial increased incidence of SRM (diameter ≤4 cm). While modern ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) techniques have high sensitivity for detecting SRM, none is able to accurately and reliably characterize them in terms of histological features. This is currently of key importance in guiding clinical decision-making in some situations, and in these cases renal biopsy should be considered. In this review, we aim to summarize the technique, diagnostic performance, and predicting factors of nondiagnostic biopsy, as well as the future perspectives.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.