Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression.

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.

Analysis of viral- and tumor antigen-specific CD4 T cell responses in healthy donors and melanoma patients

Résumé Des tentatives pour développer des traitements anti-cancéreux basés sur l'utilisation d'antigènes tumoraux ont commencé il y a plus de 10 ans. Depuis quelques années, un certain intérêt s'est portée sur une sous-population particulière des cellules du système immunitaire, les lymphocytes T CD4. Ces cellules jouent un rôle central dans les réponses immunitaires tant contre les virus que contre les cellules tumorales. Comme d'autres lymphocytes T, ces cellules sont activées de manière spécifique en reconnaissant un morceau d'antigène, appelé peptide. Ces peptides proviennent soit de protéines des cellules de l'hôte, soit des protéines étrangères (virus ou bactéries) soit de cellules transformées (cellules tumorales) et sont présentés aux lymphocytes T par des molécules du soi appelées CMH (complexe majeur d'histocompatibilité). Dans le cas des lymphocytes T CD4, ces molécules sont plus précisément des molécules du CMH de classe II (CMH II). Mis à part l'intérêt porté aux réponses médiées par les lymphocytes T cytotoxiques, un intérêt croissant pour les lymphocytes T CD4 s'est développé à cause de la place centrale qu'occupent ces cellules dans les réponses immunitaires. L'identification d'épitopes présentés par des molécules du CMH de classe II dérivés d'un grand nombre d'antigènes tumoraux, ainsi que le développement de techniques permettant de suivre les réponses immunitaires, offre des opportunités pour étudier de manière quantitative et qualitative les lymphocytes T CD4 spécifiques pour un antigène particulier chez des patients cancéreux. De plus, ces épitopes permettent d'induire des réponses médiées par les lymphocytes T CD4 et CD8 chez ces mêmes patients. Dans ce travail, notre premier but était de valider l'utilisation de multimères formés par des complexes peptide:molécules de CMH de class II (pCMH II) pour quantifier la réponse des cellules T CD4 dirigée contre l'épitope HA307-319 dérivé de la protéine hémaglutinine du virus de la grippe et présenté par HLA-DRB1*0401. En analysant des échantillons provenant de volontaires sains ayant reçus un vaccin contre la grippe, nous avons pu démontrer une expansion et une activation transitoires des lymphocytes T CD4 spécifiques pour le peptide HA307-319 après vaccination. De plus, les multimères pCMH II nous ont permis d'analyser plus en détails hétérogénéité des cellules T CD4 spécifiques pour le peptide HA307-319 présents dans le sang périphérique d'individus sains. Par la suite, notre but a été d'analyser les réponses des lymphocytes T CD4 spécifiques pour l'antigène Melan-A chez des patients atteints de mélanome métastatique. Nous avons tout d'abord démontré la présence de cellules T CD4 spécifiques pour l'épitope Melan-A51-73, présenté par HLA-DRBl*0401, qui avait déjà été préalablement décrit. Ensuite, nous avons décrit et caractérisé 2 nouveaux peptides issus de Melan-A qui sont présentés aux cellules T CD4 par différentes molécules du CMH de clans II. Des cellules spécifiques pour ces deux épitopes ont été trouvées chez 9/ 16 patients analysés. De plus, des multimères pCMH II chargés avec un des épitopes nous ont permis de détecter ex vivo des lymphocytes T CD4 spécifiques pour Melan-A dans le sang périphérique d'un patient atteint de mélanome. Mis ensemble, tous ces résultats suggèrent une potentielle utilisation des multimères pCMH II pour analyser en détail les lymphocytes T CD4 spécifiques d'antigènes définis. Cependant, le suivi ex vivo de telles cellules ne semble être possible que dans des cas bien particuliers. Néanmoins, les nouveaux épitopes issus de Melan-A et présentés par des molécules du CMH de classe II que nous avons décrits dans cette étude aideront à étudier plus en détails les lymphocytes T CD4 spécifiques pour Melan-A chez des patients atteints de mélanome, un sujet d'étude sur lequel peu de résultats sont à ce jour disponibles. Summary Attempts to develop cancer vaccines based on molecularly defined tumorassociated antigens were initiated more than 10 years ago. Apart from CTLmediated anti-tumor immunity, interests are. now focused on CD4 T cells that are central players of immune responses. The identification of MHC class-II-restricted epitopes from numerous tumor antigens together with the development of monitoring tools offers the opportunity to quantitatively and qualitatively study antigen-specific CD4 T lymphocytes in cancer patients and to induce both CTL and T helper responses in cancer patients. In this work, we first aimed at validating the use of peptide:MHC class II complex (pMHC II) multimers to quantitate the CD4 T cell response against the hemagglutinin-derived epitope HAso~-si9 from influenza virus presented by HLA-DRBl*0401. By analysing samples from healthy volunteers vaccinated with ananti-influenza vaccine, we could demonstrate a transient expansion and activation of HA-specific CD4 T cells after treatment. Moreover, pMHC II multimers helped us to study the heterogeneity of HAspecific CD4 T cells found in peripheral blood of healthy individuals. Then, we aimed to analyse Melan-A-specific CD4 T cell responses in metastatic melanoma patients. We first demonstrated the presence of CD4 T cells specific for the previously described Melan-A51_73 epitope presented by HLA-DRB 1 *0401 in peripheral blood of those patients. Second, we described and characterised 2 new Melan-A-derived peptides that are presented by different MHC II molecules to CD4 T cells. Specific cells for these epitopes were found in 9/ 16 rnelánoma patients analysed. In addition, pMHC II multimers loaded with one of the two epitopes allowed us to detect ex vivo Melan-A-specific CD4 T cells in peripheral blood of a melanoma patient. Together, these results suggest a potential use of pMHC II multimers in analysing in detail antigen-specific CD4 T cells. However, ex vivo monitoring of such cells will be possible only in particular conditions. Nevertheless, the new Melan-A-derived MHC II-restricted epitopes described here will help to study in more detail Melan-A-specific CD4 T cells in melanoma patients, a field where only scarce data are available.

The relationship between a short measure of health status and physical activity in a workplace population.

Many interventions promoting physical activity (PA) are effective in preventing disease onset, and although studies have found a positive relationship between health-related quality of life (HRQL) and PA, most of these studies have focused on older adults and those with chronic conditions. Less is known regarding the association between PA level and HRQL among healthy adults. Our objective was to analyse the relationship between PA level and HRQL among a sample of 573 employees aged 20-68 taking part in a workplace intervention to promote PA. Measures included HRQL (using a single item) and PA (i.e. Godin Leisure-Time Questionnaire). The Modified Canadian Aerobic Fitness Test (MCAFT) was also completed by 10% of the employees. MET-minute scores (assessing energy expenditure over one week) were compared across HRQL categories using ANOVA. A multiple linear regression analysis was conducted to further examine the relationship between HRQL and PA, controlling for potential covariates. Participants in the higher health status categories were found to report higher levels of energy expenditure (one-way ANOVA, p < 0.001). In the multiple linear regression model, each unit increase in health status level translated in a mean increase of 356 MET-minutes in energy expenditure (p < 0.001). This single-item assessment of health status explained six percent of the variance in energy expenditure. The study concludes that higher energy expenditure through PA among an adult workplace population is positively associated with increased health status, and it also suggests that a single-item HRQL measure is suitable for community- and population-based studies, reducing response burden and research costs.

Increased carotid intima-media thickness in the absence of atherosclerotic plaques in an adult population with Fabry disease.

AIM: Fabry disease is considered primarily as a progressive small vessel disease, with ischaemic degenerative lesions involving the kidneys, brain and heart. Macrovascular involvement in male patients includes an accelerated wall hypertrophy of the radial artery and a thickening of the intima-media of the common carotid artery. The aim of this study is to evaluate the prevalence and severity of carotid artery atherosclerosis in hemizygous and heterozygous patients with Fabry disease, compared with a matched control population. METHODS: The common carotid artery intima-media thickness (IMT) of 53 patients with Fabry disease (24 men, 29 women) was measured by high-definition ultrasonography, and the presence or absence of atherosclerotic plaques reported. Results were compared with those of 120 age-matched healthy individuals (83 men, 37 women). RESULTS: The common carotid artery IMT was increased to the same extent in male and female patients with Fabry disease (706+/-211 microm and 749+/-395 microm, respectively) compared with that of the control population (614+/-113 microm). In the Fabry population, IMT did not correlate with either systolic blood pressure or with renal function (plasma creatinine). In the control population, only systolic blood pressure was positively and significantly correlated with IMT. Atherosclerotic plaques in the common carotid artery were not observed in any patient with Fabry disease, whereas 34% of the control population had carotid artery plaques, as evidenced by focal non-homogeneous intima-media thickening greater than 1.2 mm. CONCLUSION: This study presents evidence of a major increase in common carotid artery IMT, both in hemizygous and heterozygous patients with Fabry disease, in the absence of focal atherosclerotic plaques. These results suggest that the conduit arteries may be protected from atherosclerosis in Fabry disease.

The association between inflammatory biomarkers and metabolically healthy obesity depends of the definition used.

BACKGROUND/OBJECTIVES: To assess the distribution of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) according to the different definitions of metabolically healthy obesity (MHO). SUBJECTS/METHODS: A total of 881 obese (body mass index (BMI) > or =30 kg/m2) subjects derived from the population-based CoLaus Study participated in this study. MHO was defined using six sets of criteria including different combinations of waist, blood pressure, total high-density lipoprotein cholesterol or low-density lipoprotein -cholesterol, triglycerides, fasting glucose, homeostasis model, high-sensitivity CRP, and personal history of cardiovascular, respiratory or metabolic diseases. IL-1β, IL-6 and TNF-α were assessed by multiplexed flow cytometric assay. CRP was assessed by immunoassay. RESULTS: On bivariate analysis some, but not all, definitions of MHO led to significantly lower levels of IL-6, TNF-α and CRP compared with non-MH obese subjects. Most of these differences became nonsignificant after multivariate analysis. An posteriori analysis showed a statistical power between 9 and 79%, depending on the inflammatory biomarker and MHO definition considered. Further increasing sample size to overweight+obese individuals (BMI > or =25 kg/m2, n=2917) showed metabolically healthy status to be significantly associated with lower levels of CRP, while no association was found for IL-1β. Significantly lower IL-6 and TNF-α levels were also found with some but not all MHO definitions, the differences in IL-6 becoming nonsignificant after adjusting for abdominal obesity or percent body fat. CONCLUSIONS: MHO individuals present with decreased levels of CRP and, depending on MHO definition, also with decreased levels in IL-6 and TNF-α. Conversely, no association with IL-1β levels was found.

Resting heart rate and the risk of heart failure in healthy adults: the rotterdam study.

Background- An elevated resting heart rate is associated with rehospitalization for heart failure and is a modifiable risk factor in heart failure patients. We aimed to examine the association between resting heart rate and incident heart failure in a population-based cohort study of healthy adults without pre-existing overt heart disease. Methods and Results- We studied 4768 men and women aged ≥55 years from the population-based Rotterdam Study. We excluded participants with prevalent heart failure, coronary heart disease, pacemaker, atrial fibrillation, atrioventricular block, and those using β-blockers or calcium channel blockers. We used extended Cox models allowing for time-dependent variation of resting heart rate along follow-up. During a median of 14.6 years of follow-up, 656 participants developed heart failure. The risk of heart failure was higher in men with higher resting heart rate. For each increment of 10 beats per minute, the multivariable adjusted hazard ratios in men were 1.16 (95% confidence interval, 1.05-1.28; P=0.005) in the time-fixed heart rate model and 1.13 (95% confidence interval, 1.02-1.25; P=0.017) in the time-dependent heart rate model. The association could not be demonstrated in women (P for interaction=0.004). Censoring participants for incident coronary heart disease or using time-dependent models to account for the use of β-blockers or calcium channel blockers during follow-up did not alter the results. Conclusions- Baseline or persistent higher resting heart rate is an independent risk factor for the development of heart failure in healthy older men in the general population.

Prospective monitoring of a CD4+ CD25high CD45RO+ CD127high T-cell population after first kidney transplantation following thymoglobulin or basiliximab induction

Background: Recent data have suggested that a population of CD4+ CD25high T cells, phenotypically characterized by the expression of CD45RO and CD127, is significantly expanded in stable liver and kidney transplant recipients and represents alloreactive T cells. Induction therapies may have an impact on this alloreactive T cell population. In this study, we prospectively analyzed CD4+ CD25high CD45RO+ CD127high T cells after induction with either thymoglobulin or basiliximab. Patients and methods: A total of twenty-seven kidney transplant recipients were prospectively enrolled; 14 received thymoglobulin induction followed by a 4-day course of steroids with tacrolimus and mycophenolate mofetil («thymo group»), and 13 received basiliximab induction followed by standard triple immunosuppression (tacrolimus, mycophenolate mofetil and prednisone) («BSX group»). Phenotypical analysis by flow cytometry of the expression of CD25, CD45RO and CD127 on peripheral CD4+ T cells was performed at 0, 3 and 6 months after transplantation. Twenty-four healthy subjects (HS) were studied as controls. Results: There were no differences in baseline characteristics between the groups; at 6 months, patient survival (100%), graft survival (100%), serum creatinine (thymo group versus BSX group: 129 versus 125 micromol/l) and acute rejection (2/14 versus 2/13) were not significantly different. Thymo induction produced a prolonged CD4 T cell depletion. As compared to pre-transplantation values, an expansion of the alloreactive T cell population was observed at 3 months in both thymo (mean: from 6.38% to 14.72%) and BSX (mean: from 8.01% to 18.42%) groups. At 6 months, the alloreactive T cell population remained significantly expanded in the thymo group (16.92 ± 2.87%) whereas it tended to decrease in the BSX group (10.22 ± 1.38%). Conclusion: Overall, our results indicate that the expansion of alloreactive T cells occurs rapidly after transplantation in patients receiving either thymo or BSX induction. Whether differences at later timepoints or whether different IS regimens may modify this alloreactive population remains to be studied.

Expansion and tissue infiltration of an allospecific CD4+CD25+CD45RO+IL-7Ralphahigh cell population in solid organ transplant recipients.

It has been recently shown (Seddiki, N., B. Santner-Nanan, J. Martinson, J. Zaunders, S. Sasson, A. Landay, M. Solomon, W. Selby, S.I. Alexander, R. Nanan, et al. 2006. J. Exp. Med. 203:1693-1700.) that the expression of interleukin (IL) 7 receptor (R) alpha discriminates between two distinct CD4 T cell populations, both characterized by the expression of CD25, i.e. CD4 regulatory T (T reg) cells and activated CD4 T cells. T reg cells express low levels of IL-7Ralpha, whereas activated CD4 T cells are characterized by the expression of IL-7Ralpha(high). We have investigated the distribution of these two CD4 T cell populations in 36 subjects after liver and kidney transplantation and in 45 healthy subjects. According to a previous study (Demirkiran, A., A. Kok, J. Kwekkeboom, H.J. Metselaar, H.W. Tilanus, and L.J. van der Laan. 2005. Transplant. Proc. 37:1194-1196.), we observed that the T reg CD25(+)CD45RO(+)IL-7Ralpha(low) cell population was reduced in transplant recipients (P < 0.00001). Interestingly, the CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population was significantly increased in stable transplant recipients compared with healthy subjects (P < 0.00001), and the expansion of this cell population was even greater in patients with documented humoral chronic rejection compared with stable transplant recipients (P < 0.0001). The expanded CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population contained allospecific CD4 T cells and secreted effector cytokines such as tumor necrosis factor alpha and interferon gamma, thus potentially contributing to the mechanisms of chronic rejection. More importantly, CD4(+)IL-7Ralpha(+)and CD25(+)IL-7Ralpha(+) cells were part of the T cell population infiltrating the allograft of patients with a documented diagnosis of chronic humoral rejection. These results indicate that the CD4(+)CD25(+)IL-7Ralpha(+) cell population may represent a valuable, sensitive, and specific marker to monitor allospecific CD4 T cell responses both in blood and in tissues after organ transplantation.

Iowa Medicaid Congestive Heart Failure Population Disease Management Demonstration, October 2006

Results from the Iowa Medicaid congestive heart failure population disease management demonstration confirm that a population and technology based remote monitoring platform can greatly reduce the need for costly acute care services by involving patients in their care, improving care effectiveness and promoting healthy behaviors.

Who eats healthily? A population-based study among young Swiss residents.

OBJECTIVE: To assess whether Swiss residents aged 15-24 years follow current nutritional guidelines and whether differences exist according to gender and weight status. DESIGN: Cross-sectional national survey. SETTING: Switzerland. SUBJECTS: The 1786 participants (48·4 % women) were divided into overweight, normal weight and underweight. We used traditional BMI cut-offs for people ≥18 years of age (underweight = BMI < 18·5 kg/m2, normal weight = BMI ≥ 18·5 kg/m2 and <25 kg/m2, overweight = BMI ≥ 25 kg/m2) and age- and gender-appropriate tables for people aged <18 years, with BMI calculated from self-reported weight and height. We performed bivariate analyses by gender, and then bivariate and multivariate analyses comparing overweight to normal weight people (excluding underweight, n 129, 71·6 % women) regarding adherence to recommendations for fruit, vegetables, meat, fish and dairy products; physical activity; attitude towards body weight; depression, smoking and alcohol consumption. RESULTS: Overall, adherence to nutritional guidelines was low, particularly for vegetables and dairy products. Women had a higher adherence than men except for fish and dairy products. In the multivariate analyses, overweight women had a lower vegetable intake, were less satisfied with body weight and had more often been on a diet, whereas overweight men were less satisfied with body weight and wanted to lose weight more often than their normal weight peers. There were no significant differences for physical activity. CONCLUSIONS: Overweight prevention programmes should target youth specifically by gender and promote an appropriate self-perception. Overweight women should be encouraged to eat more vegetables and men to be more sensitised on healthy food. Further research is needed to assess how to make nutritional guidelines more adaptable to young people's daily life.

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus Study).

OBJECTIVE: to assess the levels and determinants of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-a and C-reactive protein (CRP) in a healthy Caucasian population.METHODS: population sample of 2884 men and 3201 women aged 35 to 75. IL-1ß, IL-6 and TNF-a were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.RESULTS: Spearman rank correlations between duplicate cytokine measurements (N?=?80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1ß, IL-6 and TNF-a levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48-3.90) pg/ml for IL-1ß; 1.47 (0.71-3.53) pg/ml for IL-6; 2.89 (1.82-4.53) pg/ml for TNF-a and 1.3 (0.6-2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1ß levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-a levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.CONCLUSION: Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.

Are we living longer but less healthy? Trends in mortality and morbidity in Catalonia (Spain), 1994-2011

Evidence on trends in prevalence of disease and disability can clarify whether countries are experiencing a compression or expansion of morbidity. An expansion of morbidity as indicated by disease have appeared in Europe and other developed regions. It is likely that better treatment, preventive measures and increases in education levels have contributed to the declines in mortality and increments in life expectancy. This paper examines whether there has been an expansion of morbidity in Catalonia (Spain). It uses trends in mortality and morbidity from major causes of death and links of these with survival to provide estimates of life expectancy with and without diseases and functioning loss. We use a repeated cross-sectional health survey carried out in 1994 and 2011 for measures of morbidity; mortality information comes from the Spanish National Statistics Institute. Our findings show that at age 65 the percentage of life with disease increased from 52% to 70% for men, and from 56% to 72% for women; the expectation of life unable to function increased from 24% to 30% for men and 40% to 47% for women between 1994 and 2011. These changes were attributable to increases in the prevalences of diseases and moderate functional limitation. Overall, we find an expansion of morbidity along the period. Increasing survival among people with diseases can lead to a higher prevalence of diseases in the older population. Higher prevalence of health problems can lead to greater pressure on the health care system and a growing burden of disease for individuals.

Association of statins with inflammatory cytokines: a population-based Colaus study.

PURPOSE: A pleiotropic effect of statins has been reported in numerous studies. However, the association between statin use and inflammatory cytokines is controversial. We examined the associations between statin use and C-reactive protein (CRP), tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a healthy Caucasian population. METHODS: Cross-sectional study of 6184 participants aged 35-75years from Lausanne, Switzerland. Cytokines were assessed by multiplexed particle-based flow cytometric assay. Self-reported history of medication was collected for statins and other medication. 99 participants without cytokine data were excluded. RESULTS: Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. On multivariate analysis adjusting for age, gender, smoking status, body mass index, hypertension, diabetes, baseline cardiovascular disease, total cholesterol, anti-inflammatory use, other cytokine modifying drugs and other drugs, participants on statins had significantly lower CRP levels (adjusted mean±standard error: 1.22±1.05 vs. 1.38±1.04mg/L for use and non-use, respectively, p<0.01 on log-transformed data). Conversely, no association was found between statin use and IL-1β (p=0.91), IL-6 (p=0.25) or TNF-α (p=0.28) levels. On multivariate analysis, individuals in the statin group (β coefficient=-0.12; 95% CI=-0.21, -0.03) had lower levels of CRP as compared to those in the reference group (i.e. those not using statin). However, no significant associations were observed between IL-1β, IL-6 and TNF-α and statins. CONCLUSION: Individuals on statins have lower CRP levels; conversely, no effect was found for IL-1β, IL-6 and TNF-α levels.

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Human CD8 T-cell responses to Epstein-Barr virus and Cytomegalovirus in healthy donors and melanoma patients

Summary The mechanisms regulating the protective immune T-cell responses generated against the persistent Epstein-Barr virus (EBV) and Cytomegaloviru_s (CNIV) remain poorly understood. We analyzed the dynamics of cellular differentiation and T-cell receptor (TCR) clonotype selection of EBV- and CMV-specific T-cells in healthy adults and melanoma patients. While these responses could be subdivided into four T lymphocyte populations, théir proportions varied between EBV and CMV specific responses. Phenotypic and TCR clonotypic analyses supported a linear model of differentiation from the early-differentiated (EM/CD28pos) subset to the late-differentiatdc (EMRA/CD28neg) subset. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, as some clonotypes were selected with differentiation, while others were not. Latedifferentiated CMV-specific clonotypes were mostly characterized by TCRs with lower dependency on CD8 co-receptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of four years. This work was extended to the study of EBV-specific CD8 T-cell responses in melanoma patients undergoing transient lymphodepletion, followed by adoptive cell transfer (ACT) and immune reconstitution for thè treatment of their tumors. Following treatment regimen, we first observed an increase in the proportion of virus-specific T-cells in 3 out of 5 patients, accompanied by a more differentiated phenotype (EMRA/CD28neg), compared to specific cells of healthy individuals. Yet, similarly to healthy donors, clonotype selection and composition of virus-specific T-cells varied along the pathway of cellular differentiation, with some clonotypes being selected with differentiation, while others were not. Intriguingly, no novel clonotypes emerged following transient immuno-suppression and homeostatic proliferation, finding which was subsequently explained by the absence of EBV reactivation. The distribution of each clonotype within early- and late-differentiated T-cell subsets in 4 out 5 patients was highly stable over time, with those clonotypes initially found before the start of treatment that were again present at specific differentiation stages after transient lymphodepletion and ACT. These findings uncover novel features of the highly sophisticated control of steady state protective T-cell immune responses against persistent herpesviruses in healthy adults. Furthermore they reveal the striking stability of these responses in terms of clonotype selection and composition with T-cell differentiation even in situations where the immune system has been. challenged. Résumé : Les mécanismes qui régulent les réponses immunitaires de type protectrices, générées contre les virus chroniquement persistants tels que l'Epstein-Barr (EBV) ou le Cytomegalo (CMV) restent largement inconnus. Nous avons analysé la différenciation des lymphocytes T spécifiques pour ces virus, ainsi que la composition des clonotypes T (par leur récepteur T) chez les donneurs sains. Les réponses immunes peuvent être classifiées en quatre souspopulations majeures de lymphocytes T, cependant, leur proportion varie entre les réponses spécifiques contre EBV ou CMV. Ces analyses soutiennent le modèle linéaire de différenciation, à partir de la population non différenciée (EM/CD28pos) vers la population plus différenciée (ENIIZA/CD28neg). De plus, nos données sur la composition clonale de ces cellules T spécifiques ont révélé des répertoires TCR restreints, pour la réponse anti-CMV, et relativement diversifiés contre EBV. Tous les clonotypes spécifiques de ces virus identifiés dans la sous-population différenciée EMRA/CD28neg, ont également été retrouvés dans la population de cellules "mémoires". Toutefois, de fortes différences ont été observées dans les schémas de domination de ces sous-populations, en effet, certains clonotypes étaient sélectionnés avec la différenciation, alors que d'autres ne l'étaient pas. Nous avons également démontré que ces clonotypes différenciés et spécifiques pour le CMV sont caractérisés par des TCRs à faible dépendance en regard de la coopération du corécepteur CD8. Néanmoins, tous les clonotypes affichent une avidité fonctionnelle similaire, suggérant un rôle compensatoire du CD8, dans le cas des clonotypes avec une faible avidité du TCR En définitive, la composition et la sélection des clonotypes spécifiques pour chaque virus et pour chaque sous-population suit un schéma de différenciation hautement conservé au cours du temps, avec la présence de ces mêmes clonotypes au même stade de différenciation sur une période de quatre ans. Ce travail a été étendu à l'étude des réponses T CD8+ spécifiques pour le virus EBV chez les patients atteints de mélanome et recevant dans le cadre du traitement de leurs tumeurs une lymphodéplétion transitoire, suivie d'un transfert adoptif de cellules et d'une reconstitution immunitaire. Au cours de cette thérapie, nous avons en premier lieu observé pour 3 des 5 patients une augmentation de la proportion de cellules T spécifiques pour le virus, accompagné d'un phénotype plus différencié (EMRA/CD28neg), et ceci comparativement à des cellules spécifiques d'individus sains. Pourtant, comme nous l'avons observé chez les donneurs sains, la sélection et la composition des clonotypes T spécifiques varient tout au long de la différenciation cellulaire, avec certains clonotypes sélectionnés et d'autres qui ne le sont pas. Étonnamment, aucun nouveau clonotype n'a émergé après l'immuno-suppression transitoire et la prolifération homéostatique. Cette observation trouve son explication par une absence de réactivation du virus EBV chez ces patients, et ce malgré leur traitement. De plus, la distribution de chaque clonotype parmi ces sous-populations non-différenciées et différenciées reste stable au cours du traitement. Ainsi, les mêmes clonotypes initialement identifiés avant le début du traitement sont présents aux mêmes stades de différenciation après la lymphodéplétion et la prolifération homéostatique. Ces résultats ont permis d'identifier de nouveaux mécanismes impliqués dans la régulation hautement «sophistiquée » des réponses immunitaires T contre les virus persistants EBV et CMV chez les donneurs sains. En particulier, ils révèlent la grande stabilité de ces réponses en termes de sélection et de composition des clonotypes avec la différenciation cellulaire, et ce dans les situations chroniques, ainsi que dans les situations dans lesquelles le système immunitaire a été profondément perturbé.