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1.  The number of CCR5 expressing CD4+ T lymphocytes is lower in HIV-infected long-term non-progressors with viral control compared to normal progressors: a cross-sectional study 
BMC Infectious Diseases  2014;14(1):683.
Background
The HIV co-receptors CXCR4 and CCR5 play an important role in HIV infection and replication. Therefore we hypothesize that long-term non-progressors (LTNP) with viral control have lower expression of CCR5 and CXCR4 on CD4+ cells, specifically on memory T-lymphocytes since they are the primary target cells of HIV.
Methods
In this cross-sectional study, we included five HIV-infected LTNP with viral control (CD4 > 750 cell/μl & HIV < 50 copies for ≥2 years), thirteen HIV-infected and seven HIV-uninfected individuals at Radboud UMC Nijmegen, the Netherlands. We determined the CCR5 and CXCR4 expression among CD4+ and CD8+ lymphocyte subsets; memory (CD45RO+), naïve (CD45RA+) cells and regulatory T-cells (CD4+CD25highFoxP3+). In addition, CCR5∆32 polymorphism is related with disease progression and was therefore determined using polymerase chain reaction.
Results
The percentage of CCR5-expressing CD4+ cells of LTNP was comparable with healthy controls; whereas HIV-infected individuals showed more CCR5-expressing cells. This was observed in memory and naïve CD4+ cells, but not in regulatory T-cells. The mean fluorescence intensity of CCR5-expressing CD4+ cells was similar in all groups. All groups had comparable percentages of CXCR4-expressing cells. The mean fluorescence intensity of CXCR4-expressing cells was significantly higher in HIV-infected normally progressors in both memory and naïve CD4+ cells, but not in CD8+ cells. The CCR5∆32 polymorphism was not related to group.
Conclusions
We show that HIV affects -directly or indirectly- the expression of CCR5 in CD4+ T-lymphocytes; yet this effect is not seen in LTNP with viral control. Avoiding upregulation of CCR5 could be an important method via which LTNP counteracts the effects of HIV and suppresses viral replication. Exploring how LTNP suppress the upregulation of CCR5 could be an important step for discovering new therapeutics.
doi:10.1186/s12879-014-0683-0
PMCID: PMC4271479  PMID: 25495598
HIV; CCR5; CXCR4; Elite controllers; T-lymphocytes; Regulatory T-cells; Memory T cells; Naïve T cells; HIV co-receptors
2.  Longitudinal Analysis of T and B Cell Phenotype and Function in Renal Transplant Recipients with or without Rituximab Induction Therapy 
PLoS ONE  2014;9(11):e112658.
Background
Prevention of rejection after renal transplantation requires treatment with immunosuppressive drugs. Data on their in vivo effects on T- and B-cell phenotype and function are limited.
Methods
In a randomized double-blind placebo-controlled study to prevent renal allograft rejection, patients were treated with tacrolimus, mycophenolate mofetil (MMF), steroids, and a single dose of rituximab or placebo during transplant surgery. In a subset of patients, we analyzed the number and phenotype of peripheral T and B cells by multiparameter flow cytometry before transplantation, and at 3, 6, 12, and 24 months after transplantation.
Results
In patients treated with tacrolimus/MMF/steroids the proportion of central memory CD4+ and CD8+ T cells was higher at 3 months post-transplant compared to pre-transplant levels. In addition, the ratio between the percentage of central memory CD4+ and CD4+ regulatory T cells was significantly higher up to 24 months post-transplant compared to pre-transplant levels. Interestingly, treatment with tacrolimus/MMF/steroids resulted in a shift toward a more memory-like B-cell phenotype post-transplant. Addition of a single dose of rituximab resulted in a long-lasting B-cell depletion. At 12 months post-transplant, the small fraction of repopulated B cells consisted of a high percentage of transitional B cells. Rituximab treatment had no effect on the T-cell phenotype and function post-transplant.
Conclusions
Renal transplant recipients treated with tacrolimus/MMF/steroids show an altered memory T and B-cell compartment post-transplant. Additional B-cell depletion by rituximab leads to a relative increase of transitional and memory-like B cells, without affecting T-cell phenotype and function.
Trial Registration
ClinicalTrials.gov NCT00565331
doi:10.1371/journal.pone.0112658
PMCID: PMC4231065  PMID: 25393622
3.  Phosphatidylserine exposure on stored red blood cells as a parameter for donor-dependent variation in product quality 
Blood Transfusion  2014;12(2):204-209.
Background
Exposure of phosphatidylserine on the outside of red blood cells contributes to recognition and removal of old and damaged cells. The fraction of phosphatidylserine-exposing red blood cells varies between donors, and increases in red blood cell concentrates during storage. The susceptibility of red blood cells to stress-induced phosphatidylserine exposure increases with storage. Phosphatidylserine exposure may, therefore, constitute a link between donor variation and the quality of red blood cell concentrates.
Materials and methods
In order to examine the relationship between storage parameters and donor characteristics, the percentage of phosphatidylserine-exposing red blood cells was measured in red blood cell concentrates during storage and in fresh red blood cells from blood bank donors. The percentage of phosphatidylserine-exposing red blood cells was compared with red blood cell susceptibility to osmotic stress-induced phosphatidylserine exposure in vitro, with the regular red blood cell concentrate quality parameters, and with the donor characteristics age, body mass index, haemoglobin level, gender and blood group.
Results
Phosphatidylserine exposure varies between donors, both on red blood cells freshly isolated from the blood, and on red blood cells in red blood cell concentrates. Phosphatidylserine exposure increases with storage time, and is correlated with stress-induced phosphatidylserine exposure. Increased phosphatidylserine exposure during storage was found to be associated with haemolysis and vesicle concentration in red blood cell concentrates. The percentage of phosphatidylserine-exposing red blood cells showed a positive correlation with the plasma haemoglobin concentration of the donor.
Discussion
The fraction of phosphatidylserine-exposing red blood cells is a parameter of red blood cell integrity in red blood cell concentrates and may be an indicator of red blood cell survival after transfusion. Measurement of phosphatidylserine exposure may be useful in the selection of donors and red blood cell concentrates for specific groups of patients.
doi:10.2450/2013.0106-13
PMCID: PMC4039702  PMID: 24120596
aging; RBC; phosphatidylserine; storage; donor
4.  Selective expansion of human natural killer cells leads to enhanced alloreactivity 
Cellular and Molecular Immunology  2013;11(2):160-168.
In allogeneic stem cell transplantation (SCT), natural killer (NK) cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease (GVHD). This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors (KIRs) within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 (C1) or Cw4 (C2) transfected K562 stimulator cells. Coculture of KIR+ NK cells with C1 or C2 positive K562 cells, in the presence of IL-2+IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR+ NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107+ cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.
doi:10.1038/cmi.2013.56
PMCID: PMC4003378  PMID: 24240123
alloreactivity; cytotoxicity; immunotherapy; KIR; NK cells; stem cell transplantation
5.  Redefining Strategies to Introduce Tolerance-Inducing Cellular Therapy in Human beings to Combat Autoimmunity and Transplantation Reactions 
Clinical translation of tolerance-inducing cell therapies requires a novel approach focused on innovative networks, patient involvement, and, foremost, a fundamental paradigm shift in thinking from both Academia, and Industry and Regulatory Agencies. Tolerance-inducing cell products differ essentially from conventional drugs. They are personalized and target interactive immunological networks to shift the balance toward tolerance. The human cell products are often absent or fundamentally different in animals. This creates important limitations of pre-clinical animal testing for safety and efficacy of these products and calls for novel translational approaches, which require the combined efforts of the different parties involved. Dedicated international and multidisciplinary consortia that focus on clinical translation are of utmost importance. They can help in informing and educating regulatory policy makers on the unique requirements for these cell products, ranging from pre-clinical studies in animals to in vitro human studies. In addition, they can promote reliable immunomonitoring tools. The development of tolerance-inducing cell products requires not only bench-to-bedside but also reverse translation, from bedside back to the bench.
doi:10.3389/fimmu.2014.00392
PMCID: PMC4133652  PMID: 25177323
tolerance; regulatory T cells; dendritic cells; regulatory agencies; autoimmunity; transplantation
6.  Immune responses to stress after stress management training in patients with rheumatoid arthritis 
Arthritis Research & Therapy  2013;15(6):R200.
Introduction
Psychological stress may alter immune function by activating physiological stress pathways. Building on our previous study, in which we report that stress management training led to an altered self-reported and cortisol response to psychological stress in patients with rheumatoid arthritis (RA), we explored the effects of this stress management intervention on the immune response to a psychological stress task in patients with RA.
Methods
In this study, 74 patients with RA, who were randomly assigned to either a control group or a group that received short stress management training, performed the Trier Social Stress Test (TSST) 1 week after the intervention and at a 9-week follow-up. Stress-induced changes in levels of key cytokines involved in stress and inflammatory processes (for example, interleukin (IL)-6 and IL-8) were assessed.
Results
Basal and stress-induced cytokine levels were not significantly different in patients in the intervention and control groups one week after treatment, but stress-induced IL-8 levels were lower in patients in the intervention group than in the control group at the follow-up assessment.
Conclusions
In line with our previous findings of lower stress-induced cortisol levels at the follow-up of stress management intervention, this is the first study to show that relatively short stress management training might also alter stress-induced IL-8 levels in patients with RA. These results might help to determine the role of immunological mediators in stress and disease.
Trial registration
The Netherlands National Trial Register (NTR1193)
doi:10.1186/ar4390
PMCID: PMC3978719  PMID: 24274618
7.  Rho Kinase Inhibitor Y-27632 Prolongs the Life Span of Adult Human Keratinocytes, Enhances Skin Equivalent Development, and Facilitates Lentiviral Transduction 
Tissue Engineering. Part A  2012;18(17-18):1827-1836.
The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.
doi:10.1089/ten.tea.2011.0616
PMCID: PMC3432898  PMID: 22519508
8.  Iron Status and Systemic Inflammation, but Not Gut Inflammation, Strongly Predict Gender-Specific Concentrations of Serum Hepcidin in Infants in Rural Kenya 
PLoS ONE  2013;8(2):e57513.
Hepcidin regulation by competing stimuli such as infection and iron deficiency has not been studied in infants and it’s yet unknown whether hepcidin regulatory pathways are fully functional in infants. In this cross-sectional study including 339 Kenyan infants aged 6.0±1.1 months (mean±SD), we assessed serum hepcidin-25, biomarkers of iron status and inflammation, and fecal calprotectin. Prevalence of inflammation, anemia, and iron deficiency was 31%, 71%, 26%, respectively. Geometric mean (±SD) serum hepcidin was 6.0 (±3.4) ng/mL, and was significantly lower in males than females. Inflammation (C-reactive protein and interleukin-6) and iron status (serum ferritin, zinc protoporphyrin and soluble transferrin receptor) were significant predictors of serum hepcidin, explaining nearly 60% of its variance. There were small, but significant differences in serum hepcidin comparing iron deficient anemic (IDA) infants without inflammation to iron-deficient anemic infants with inflammation (1.2 (±4.9) vs. 3.4 (±4.9) ng/mL; P<0.001). Fecal calprotectin correlated with blood/mucus in the stool but not with hepcidin. Similarly, the gut-linked cytokines IL-12 and IL-17 did not correlate with hepcidin. We conclude that hepcidin regulatory pathways are already functional in infancy, but serum hepcidin alone may not clearly discriminate between iron-deficient anemic infants with and without infection. We propose gender-specific reference values for serum hepcidin in iron-replete infants without inflammation.
doi:10.1371/journal.pone.0057513
PMCID: PMC3583867  PMID: 23460869
9.  Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis 
Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte–mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine–mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.
doi:10.1172/JCI65642
PMCID: PMC3561798  PMID: 23348739
10.  Humanized Mouse Model of Skin Inflammation Is Characterized by Disturbed Keratinocyte Differentiation and Influx of IL-17A Producing T Cells 
PLoS ONE  2012;7(10):e45509.
Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.
As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin.
In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases.
doi:10.1371/journal.pone.0045509
PMCID: PMC3477148  PMID: 23094018
11.  Storage-Induced Changes in Erythrocyte Membrane Proteins Promote Recognition by Autoantibodies 
PLoS ONE  2012;7(8):e42250.
Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.
doi:10.1371/journal.pone.0042250
PMCID: PMC3411782  PMID: 22879923
12.  Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters 
Cancer Immunology, Immunotherapy  2012;61(11):2003-2011.
Purpose
Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.
Experimental design
Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.
Results
KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.
Conclusions
We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.
Electronic supplementary material
The online version of this article (doi:10.1007/s00262-012-1263-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00262-012-1263-z
PMCID: PMC3493659  PMID: 22527252
Adjuvant; Dendritic cell; Humoral response; Immunocompetence; Keyhole limpet hemocyanin; Vaccine
13.  Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling 
BMC Immunology  2012;13:12.
Background
T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.
Results
Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.
Conclusions
This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.
doi:10.1186/1471-2172-13-12
PMCID: PMC3355027  PMID: 22413885
Signal transduction pathways; Gene expression profiling; T lymphocytes; Th1 and Th2 development
14.  Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues 
PLoS ONE  2012;7(2):e30930.
A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.
doi:10.1371/journal.pone.0030930
PMCID: PMC3272048  PMID: 22319595
15.  Seasonal Variation in Vitamin D3 Levels Is Paralleled by Changes in the Peripheral Blood Human T Cell Compartment 
PLoS ONE  2012;7(1):e29250.
It is well-recognized that vitamin D3 has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D3 status. Here, we investigated if and to what extent seasonality of vitamin D3 levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D3 and 1,25(OH)2D3 levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naïve CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D3 status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings.
doi:10.1371/journal.pone.0029250
PMCID: PMC3250425  PMID: 22235276
16.  1,25-dihydroxyvitamin D3 Modulates Cytokine Production Induced by Candida albicans: Impact of Seasonal Variation of Immune Responses 
The Journal of Infectious Diseases  2011;203(1):122-130.
Background. Our interest in immunological effects produced by vitamin D3 (1,25(OH)2D3) and its therapeutic potential prompted us to examine the role of 1,25(OH)2D3 on cytokine production by Candida albicans.
Methods. Peripheral blood mononuclear cells (PBMC) with stimulated C. albicans and 1,25(OH)2D3, cytokine concentrations were measured in supernatant. Quantitative polymerase chain reaction (qPCR) was performed for T cell transcription factors, SOCS1 and 3. TLR2/4, Dectin-1, and mannose receptor expression was studied using flow cytometry and qPCR. An ex-vivo stimulation study was carried out in healthy volunteers to investigate the seasonality of immune response to C. albicans.
Results. Upon in vitro C. albicans stimulation, 1,25(OH)2D3 induced a dose-dependent, down-regulation of IL-6, TNFα, IL-17, and IFNγ. It also increased IL-10 production. The shift in cytokine profile was not due to 1,25(OH)2D3 augmenting expression of either Thelper differentiation factors or SOCS1 and SOCS3 mRNA. 1,25(OH)2D3 inhibited TLR2, TLR4, Dectin-1, and MR mRNA and protein expression. In our seasonality study, both IL-17 and IFNγ levels were suppressed in summer when 25(OH)D3 levels were elevated.
Conclusion. Vitamin D3 skews cytokine responses toward an antiinflammatory profile, mediated by suppression of TLR2, TLR4, Dectin-1, and MR transcription, leading to reduced surface expression. The biological relevance of these effects has been confirmed by the seasonality of cytokine responses.
doi:10.1093/infdis/jiq008
PMCID: PMC3086448  PMID: 21148505
17.  Deletion of the late cornified envelope (LCE) 3B and 3C genes as a susceptibility factor for psoriasis 
Nature genetics  2009;41(2):211-215.
Psoriasis is a common inflammatory skin disease with a prevalence of 2% to 3% in Caucasians1. In a genome-wide search for copy number variants (CNV) using a sample pooling approach we have identified a deletion comprising LCE3B and LCE3C, members of the late cornified envelope (LCE) gene cluster2. The absence of LCE3B and LCE3C (LCE3C-LCE3B-del) is significantly associated (p=1.38E-08) with risk of psoriasis in 2,831 samples from Spain, The Netherlands, Italy and the USA, and in a family-based study (p=5.4E-04). LCE3C-LCE3B-del is tagged by rs4112788 (r2=0.93), which is also strongly associated with psoriasis (p<6.6E-09). LCE3C-LCE3B-del shows epistatic effects with the HLA-Cw6 allele on the development of psoriasis in Dutch samples, and multiplicative effects in the other samples. LCE expression can be induced in normal epidermis by skin barrier disruption and is strongly expressed in psoriatic lesions, suggesting that compromised skin barrier function plays a role in psoriasis susceptibility.
doi:10.1038/ng.313
PMCID: PMC3128734  PMID: 19169253
18.  High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy 
PLoS ONE  2010;5(2):e9221.
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.
doi:10.1371/journal.pone.0009221
PMCID: PMC2821405  PMID: 20169160
19.  Clinical Grade Treg: GMP Isolation, Improvement of Purity by CD127pos Depletion, Treg Expansion, and Treg Cryopreservation 
PLoS ONE  2008;3(9):e3161.
Background
Treg based immunotherapy is of great interest to facilitate tolerance in autoimmunity and transplantation. For clinical trials, it is essential to have a clinical grade Treg isolation protocol in accordance with Good Manufacturing Practice (GMP) guidelines. To obtain sufficient Treg for immunotherapy, subsequent ex vivo expansion might be needed.
Methodology/Principal Findings
Treg were isolated from leukapheresis products by CliniMACS based GMP isolation strategies, using anti-CD25, anti-CD8 and anti-CD19 coated microbeads. CliniMACS isolation procedures led to 40–60% pure CD4posCD25highFoxP3pos Treg populations that were anergic and had moderate suppressive activity. Such CliniMACS isolated Treg populations could be expanded with maintenance of suppressive function. Alloantigen stimulated expansion caused an enrichment of alloantigen-specific Treg. Depletion of unwanted CD19pos cells during CliniMACS Treg isolation proved necessary to prevent B-cell outgrowth during expansion. CD4posCD127pos conventional T cells were the major contaminating cell type in CliniMACS isolated Treg populations. Depletion of CD127pos cells improved the purity of CD4posCD25highFoxP3pos Treg in CliniMACS isolated cell populations to approximately 90%. Expanded CD127neg CliniMACS isolated Treg populations showed very potent suppressive capacity and high FoxP3 expression. Furthermore, our data show that cryopreservation of CliniMACS isolated Treg is feasible, but that activation after thawing is necessary to restore suppressive potential.
Conclusions/Significance
The feasibility of Treg based therapy is widely accepted, provided that tailor-made clinical grade procedures for isolation and ex vivo cell handling are available. We here provide further support for this approach by showing that a high Treg purity can be reached, and that isolated cells can be cryopreserved and expanded successfully.
doi:10.1371/journal.pone.0003161
PMCID: PMC2522271  PMID: 18776930
20.  Immunological Monitoring of Renal Transplant Recipients to Predict Acute Allograft Rejection Following the Discontinuation of Tacrolimus 
PLoS ONE  2008;3(7):e2711.
Background
Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal.
Principal Findings
Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors.
Conclusions
Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced.
doi:10.1371/journal.pone.0002711
PMCID: PMC2442873  PMID: 18628993
21.  Ex Vivo Generation of Human Alloantigen-Specific Regulatory T Cells from CD4posCD25high T Cells for Immunotherapy 
PLoS ONE  2008;3(5):e2233.
Background
Regulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy.
Methodology/Principal Findings
Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4posCD25high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.
Conclusions/Significance
The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.
doi:10.1371/journal.pone.0002233
PMCID: PMC2376059  PMID: 18493605
22.  Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-G 
PLoS Biology  2005;4(1):e9.
Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy.
KIR2DL4, a human killer cell immunoglobulin receptor expressed on natural killer cells, can be stimulated by soluble antibody or the soluble version of its natural ligand (HLA-G), and may signal from within endosomes.
doi:10.1371/journal.pbio.0040009
PMCID: PMC1318474  PMID: 16366734

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