Renal graft function in transplanted patients correlates with CD45RC T cell phenotypic signature.

Biomarkers that could predict the evolution of the graft in transplanted patients and that could allow to adapt the care of the patients would be an invaluable tool. Additionally, certain biomarkers can be target of treatments and help to stratify patients. Potential effective biomarkers have been identified but still need to be confirmed. CD45RC, one of the splicing variants of the CD45 molecule, a tyrosine phosphatase that is critical in negatively or positively regulating the TCR and the BCR signaling, is one marker already described. The frequency of CD8+ T cells expressing high levels of CD45RC before transplantation is increased in patients with an increased risk of acute rejection. However, single biomarkers have limited predictive reliability and the correlation of the expression levels of CD45RC with other cell markers was not reported. In this study, we performed a fluorescent-based high dimensional immunophenotyping of T cells on a cohort of 69 kidney transplant patients either with stable graft function or having experienced acute transplant rejection during the first year after transplantation or at the time of rejection. We identified combinations of markers and cell subsets associated with activation/inflammation or Tregs/tolerance (HLA-DR, PD-1, IFNγ, CD28) as significant biomarkers associated to transplant outcome, and showed the importance of cell segregation based on the CD45RC marker to identify the signature of a stable graft function. Our study highlights potential reliable biomarkers in transplantation to predict and/or monitor easily graft-directed immune responses and adapt immunosuppression treatments to mitigate adverse effects.

CD4+ and CD8+ regulatory T cell characterization in the rat using a unique transgenic Foxp3-EGFP model.

BACKGROUND: Regulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged. RESULTS: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2. CONCLUSIONS: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.

Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells.

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

IL-34 deficiency impairs FOXP3+ Treg function in a model of autoimmune colitis and decreases immune tolerance homeostasis.

BACKGROUND: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined. METHODS: We generated Il34-/- rats and using both Il34-/- rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34-/- rats, we further analyzed the impact of the absence of expression of IL-34 for CD4+ Tregs suppressive function. We investigated the potential of IL-34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL-34 in the serum and kidney transplant rejection. RESULTS: Here we report that the absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in Il34-/- animals. Moreover, we revealed the striking inability of Il34-/- CD4+ Tregs to protect Il2rg-/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34+/+ CD4+ Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients. CONCLUSION: Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity. HIGHLIGHTS: -Absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with a production of multiple auto-antibodies and exacerbated immune pathology under inflammatory conditions. -Il34-/- CD4+ Tregs are unable to protect Il2rg-/- rats from colitis induced by transfer of pathogenic cells. -IL-34 treatment delayed EAE in mice, as well as acute GVHD and human skin allograft rejection in immune-humanized immunodeficient NSG mice.

Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome.

Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T (Tconv) cells (CD45RChi), their precursors, and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs; CD45RClo/-). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation, but its potential has not been examined in autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare genetic syndrome caused by loss-of-function mutations of autoimmune regulator (AIRE), a key central tolerance mediator, leading to abnormal autoreactive T cell responses and autoantibody production. Herein, we show that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective for both prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RChi T cells, inhibited CD45RChi B cells, and restored the Treg/Tconv cell ratio and the altered Treg transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells, and lesioned organs from APECED patients were infiltrated by CD45RChi cells. Our observations highlight the potential role for CD45RChi cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.

Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells.

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

IL-34 Actions on FOXP3+ Tregs and CD14+ Monocytes Control Human Graft Rejection.

Cytokines are major players regulating immune responses toward inflammatory and tolerogenic results. In organ and bone marrow transplantation, new reagents are needed to inhibit tissue destructive mechanisms and eventually induce immune tolerance without overall immunosuppression. IL-34 is a cytokine with no significant homology with any other cytokine but that acts preferentially through CSF-1R, as CSF-1 does, and through PTPζ and CD138. Although IL-34 and CSF-1 share actions, a detailed analysis of their effects on immune cells needs further research. We previously showed that both CD4+ and CD8+ FOXP3+ Tregs suppress effector T cells through the production of IL-34, but not CSF-1, and that this action was mediated through antigen-presenting cells. We showed here by single-cell RNAseq and cytofluorimetry that different subsets of human monocytes expressed different levels of CSF-1R, CD138, and PTPζ and that both CD4+ and CD8+ FOXP3+ Tregs expressed higher levels of CSF-1R than conventional T cells. The effects of IL-34 differed in the survival of these different subpopulations of monocytes and RNAseq analysis showed several genes differentially expressed between IL-34, CSF-1, M0, M1, and also M2 macrophages. Acute graft-vs.-host disease (aGVHD) in immunodeficient NSG mice injected with human PBMCs was decreased when treated with IL-34 in combination with an anti-CD45RC mAb that depleted conventional T cells. When IL-34-differentiated monocytes were used to expand Tregs in vitro, both CD4+ and CD8+ FOXP3+ Tregs were highly enriched and this effect was superior to the one obtained with CSF-1. Human CD8+ Tregs expanded in vitro with IL-34-differentiated allogeneic monocytes suppressed human immune responses in an NSG mouse aGVHD model humanized with hPBMCs. Overall, we showed that IL-34 induced the differentiation of human monocytes with a particular transcriptional profile and these cells favored the development of potent suppressor FOXP3+ Tregs.

Biomechanical constraints remain major risk factors for low back pain. Results from a prospective cohort study in French male employees.

BACKGROUND CONTEXT: Low back pain (LBP) is a major public health problem, with a considerable impact on workers. PURPOSE: To model the risk of LBP in the male general working population. STUDY DESIGN/SETTING: Repeated cross-sectional surveys in a wide occupational setting. PATIENT SAMPLE: A random sample of 2,161 men working in various occupations in a French region participated in a first survey in 2002, and 1,313 of these (60.8%) participated in a second survey in 2007. OUTCOME MEASURE: The self-reported prevalence of LBP during the previous week in the second survey. METHODS: Twenty-one biomechanical, organizational, psychosocial, and individual factors were assessed in the first survey. The association between these potential risk factors and the prevalence of later LBP (in the second survey) was studied, using multistep logistic regression models. RESULTS: Three hundred ninety-four men reported LBP in the second survey (prevalence 30.0%). The final multivariate model highlighted four risk factors: frequent bending (odds ratio [OR], 1.45, 95% confidence interval [CI], 1.07-1.97 for bending forward only; and OR, 2.13, 95% CI, 1.52-3.00 for bending both forward and sideways), driving industrial vehicles (OR, 1.35; 95% CI, 1.00-1.81), working more hours than officially planned (OR, 1.38; 95% CI, 1.05-1.81), and reported low support from supervisors (OR, 1.35; 95% CI, 1.02-1.79). CONCLUSIONS: These results emphasize that biomechanical factors remain worth considering, even when psychosocial factors are taken into account, and provide a significant contribution to preventive strategies.

[Difficulties of a longitudinal epidemiological follow-up study in French occupational medicine]. / Les difficultés d'un suivi epidémiologique longitudinal dans les services de santé au travail.

INTRODUCTION: This study was designed to describe the difficulties of epidemiological follow-up of employees by occupational health services. METHODS: This study was based on two transverse studies conducted by the Pays de la Loire musculoskeletal disorders (MSD) surveillance network. Eighty-three occupational health physicians included 3,710 employees between 2002 and 2005 and had to review them between 2007 and 2009. Thirteen of these physicians changed jobs and 7 changed geographical sector in the same occupational health service. RESULTS: Another 94 physicians were contacted and 85 agreed to participate in the network. The follow-up rate was 43%: 1,044 employees were reviewed by the physician who saw them on inclusion and 567 employees were reviewed by another physician of the network. The other employees were distributed as follows: 25% were lost to follow-up and their occupational status remained unknown; 23% were still employed with an identified occupational health physician but had not attended a medical visit during the follow-up study; 5% had left the occupational health surveillance system. Only 23 employees refused to participate in the follow-up and 105 employees had a physician who refused to participate. DISCUSSION: There is therefore a considerable mobility of occupational health physicians, which interfered with follow-up despite their good mobilization and a high percentage of employee are lost to follow-up after having left their jobs. More appropriate systems must be set up to follow populations of employees, such as new collaborations with general practitioners.

Work-related risk factors for incidence of lateral epicondylitis in a large working population.

OBJECTIVES: This study aims to estimate the association between repeated measures of occupational risk factors and the incidence of lateral epicondylitis in a large working population. METHODS: A total of 3710 workers in a French region were included in 2002-2005, and among them 1046 had a complete follow-up in 2007-2010. At both stages, occupational health physicians assessed the presence of lateral epicondylitis and workers self-reported their occupational exposures. Poisson models were performed to assess the incidence rate ratios (IRR) separately by sex using multiple imputed data. RESULTS: The annual incidence rate of lateral epicondylitis was estimated as 1.0 [95% confidence interval (95% CI) 0.7-1.3] per 100 workers among men and 0.9 (95% CI 0.6-1.3) among women. Workers aged >45 years had higher incidence than those aged <30 years (significant at 10%). Among men, high physical exertion combined with elbow flexion/extension or extreme wrist bending (>2 hours/day) was a risk factor, with an age-adjusted IRR of 3.2 (95% CI 1.5-6.4) for workers exposed at both questionnaires [3.3 (95% CI 1.4-7.6) among women]. CONCLUSIONS: This study highlights the importance of temporal dimensions for occupational risk factors on the incidence of lateral epicondylitis. Further research should evaluate the risk associated with the duration and repetition of occupational exposure on the incidence of lateral epicondylitis.

Employment and occupational outcomes of workers with musculoskeletal pain in a French region.

OBJECTIVES: To study the employment and occupational outcomes of workers who were diagnosed with upper limb musculoskeletal disorders (UL-MSDs) or had complained of upper limb musculoskeletal pain a few years before compared with workers who had no upper limb pain. METHODS: In 2002-2005, an epidemiological surveillance system was set up. Occupational physicians examined 3710 randomly selected workers. It focused on six UL-MSDs: rotator cuff syndrome, lateral epicondylitis, flexor-extensor peritendinitis of the hands and fingers, de Quervain's disease, carpal tunnel syndrome and ulnar tunnel syndrome. Three groups were constituted: a 'UL-MSD' group (workers with a clinically diagnosed UL-MSD at baseline, 13% of the cohort); a 'PAIN' group (workers with pain in the previous 7 days at baseline and without any clinically diagnosed form, 38%); and a 'HEALTHY' group (workers with no disorder or upper limb pain in the previous 7 days, 49%). They completed a questionnaire between 2007 and 2009. RESULTS: A total of 2332 responded. Fewer subjects were still in work in the 'UL-MSD' group (79.3%) than in the 'PAIN' (85.9%) and 'HEALTHY' (90.4%) groups, the difference remaining significant after adjusting for gender, age, occupational category, type of company and comorbidities. Of the subjects still in work, 24% had changed their work station in the same company in the 'PAIN' group compared with 19% in the 'HEALTHY' group and 21% in the 'UL-MSD' group. CONCLUSIONS: This study showed the impact of musculoskeletal pain on employment outcome and the difficulty of keeping workers with musculoskeletal problems at work.

Effects of individual and work-related factors on incidence of shoulder pain in a large working population.

OBJECTIVES: The aim of this study was to assess the effects of individual and work-related factors on the incidence of shoulder pain in a large French working population. METHODS: A total of 3,710 workers of a French region were randomly included in a cross-sectional study between 2002 and 2005. They completed a self-administered questionnaire about musculoskeletal symptoms, individual factors and exposure to work constraints. In 2007, 2,332 responded to a follow-up questionnaire. The Nordic questionnaire was used both times to assess shoulder pain during the preceding 7 days. Associations between incident shoulder pain and individual and work-related factors at baseline were studied by multivariate logistic regression for both genders. RESULTS: A total of 946 men and 709 women without shoulder pain at baseline were eligible for the analyses. At follow-up, 105 men (11.1%) and 145 women (20.5%) reported shoulder pain. For men, age (OR 3.3, 95% CI, 1.7-6.5 for ≥50 yr), working with arms above the shoulder (1.5; 1.0-2.3) and high perceived physical exertion (1.6; 1.0-2.5) increased the risk of incident shoulder pain. For women, the factors associated with incident shoulder pain were age (2.9; 1.5-5.8 for ≥50 yr), obesity (2.5; 1.4-4.5), temporary employment (2.1; 1.1-3.7), high perceived physical exertion (2.2; 1.4-3.5) and low decision latitude (1.6; 1.0-2.3). CONCLUSION: Age was the strongest predictor of incident shoulder pain in both genders. BMI and biomechanical and psychosocial factors were also identified as risk factors, whereas no factor related to work organization remained in the final models.

Risk factors for incidence of rotator cuff syndrome in a large working population.

OBJECTIVES: The aim of this study was to assess the effects of personal and work-related factors on the incidence of rotator cuff syndrome (RCS) in a large working population. METHODS: A total of 3710 French workers were included in a cross-sectional study in 2002-2005. All completed a self-administered questionnaire about personal factors and work exposure. Using a standardized physical examination, occupational physicians established a diagnosis of RCS. Between 2007-2010, 1611 workers were re-examined. Associations between RCS and risk factors at baseline were analyzed by logistic regression. RESULTS: A total of 839 men and 617 women without RCS at baseline were eligible for analysis. RCS was diagnosed in 51 men (6.1%) and 45 women (7.3%). The risk of RCS increased with age for both genders [odds ratio (OR) 4.7 (95% confidence interval [95% CI] 2.2-10.0) for men aged 45-49 years and 5.4 (95% CI 2.3-13.2) for women aged 50-59 years; reference <40 years]. For men, the work-related risk factors were repeated posture with the arms above the shoulder level combined with high perceived physical exertion [OR 3.3 (95% CI 1.3-8.4)] and low coworker support [OR 2.0 (95% CI 1.1-3.9)]. For women, working with colleagues in temporary employment [OR 2.2 (95% CI 1.2-4.2)] and repeated arm abduction (60-90°) [OR 2.6 (95% CI 1.4-5.0)] were associated with RCS. CONCLUSIONS: Age was the strongest predictor for incident cases of RCS, and arm abduction was the major work-related risk factor for both genders. Lack of social support was a predictor for RCS among men.

[Study of population profiles in relation to the level of suicide risk in France: Study "Mental health in the general population"]. / Etude des profils de population selon le niveau de risque suicidaire en France : Enquête "santé mentale en population générale".

Suicidal behaviour is a very important public health issue. The French study of mental health in the general population casts a whole new insight on this issue thanks to the size of the sample used, to its representative nature and to the variety of the collected data. This study aims at defining better the relationships between the factors of suicide risk within a noninstitutionalized adult population and more specifically between the socioeconomic and the psychopathological factors. The final aim is to help define the intervention strategies which should be developed in the context of prevention programs. The method used consists in estimating the suicide risk for each person included in the study by developing a standardized indicator. Six questions taken from the MINI (Mini International Neuropsychiatric Interview) were used to define the four levels of suicide risks which compose this indicator. Next, this indicator was matched for the socioeconomic variables of the study as well as for the main psychopathological categories. A factor analysis of the numerous relations was then carried out. Its principle consisted in synthesizing the information contained in a great number of variables and individuals thanks to the mathematical projection of these features onto a graph. The variables which were retained for the analysis were those which presented the richest relationship with the main variable.(that is to say the level of suicide risk). The estimated prevalence rate of suicidal risk in the general population (with at least one positive answer) is 13.7% which can be divided into 9.7% of low risk, 2.1% of medium risk and 1.9% of high risk. The relationship between the presence of a psychopathology and a medium or high risk of suicide is quite significant. What is more, the presence of associated pathologies (comorbidities) increases the risk. The highest prevalence of risk is observed in psychotic and depressive disorders. However, suicide risk exists in some people who do not present any detected psychopathology : the statistical analysis reveals an excessive medium and high suicide risk in relation to a low family income, unemployment, separation and the 18 to 24 age group. The multidimensional analysis brings to light several specific aspects : the principal explanation shows a relationship between unfavourable socio economic status and the presence of suicide risk at a level which is not equal to zero. The second explanatory line defines the level of risk according to the principal psychopathological characteristics. These two lines define a plane which enables to differentiate low risk groups from medium risk groups and high risk groups. The latter consists mainly in isolated pathological factors or associated factors (comorbidities). The medium and high risk groups are composed mainly of the combination of the two variables. To conclude, these results - which are necessarily flimsy since they are based on epidemiological and statistical analysis - do however match up with the data of the epidemiologic literature in an interesting way and raise the question of an intervention and prevention strategy that would integrate better the medical factors and the socio economic aspects into its program. They should be completed by targeted forward clinical studies as well as by more precise epidemiological patterns.