Search

The worldwide epidemic of overweight and obesity has led to an increase in associated metabolic comorbidities. Obesity induces chronic low-grade inflammation in white adipose tissue (WAT). However, the function and regulation of both innate and adaptive immune cells in human WAT under conditions of obesity and calorie restriction (CR) is not fully understood yet. Using a randomized interventional design, we investigated postmenopausal overweight or obese female subjects who either underwent CR for 3 mo followed by a 4-wk phase of weight maintenance or had to maintain a stable weight over the whole study period. A comprehensive immune phenotyping protocol was conducted using validated multiparameter flow cytometry analysis in blood and s.c. WAT (SAT). The TCR repertoire was analyzed by next-generation sequencing and cytokine levels were determined in SAT. Metabolic parameters were determined by hyperinsulinemic-euglycemic clamp. We found that insulin resistance correlates significantly with a shift toward the memory T cell compartment in SAT. TCR analysis revealed a diverse repertoire in SAT of overweight or obese individuals. Additionally, whereas weight loss improved systemic insulin sensitivity in the intervention group, SAT displayed no significant improvement of inflammatory parameters (cytokine levels and leukocyte subpopulations) compared with the control group. Our data demonstrate the accumulation of effector memory T cells in obese SAT and an association between systemic glucose homeostasis and inflammatory parameters in obese females. The long-standing effect of obesity-induced changes in SAT was demonstrated by preserved immune cell composition after short-term CR-induced weight loss.

In multiple sclerosis (MS), relapse rate is decreased by 70-80% in the third trimester of pregnancy. However, the underlying mechanisms driving this effect are poorly understood. Evidence suggests that CD56bright NK cell frequencies increase during pregnancy. Here, we analyze pregnancy-related NK cell shifts in a large longitudinal cohort of pregnant women with and without MS, and provide in-depth phenotyping of NK cells. In healthy pregnancy and pregnancy in MS, peripheral blood NK cells showed significant frequency shifts, notably an increase of CD56bright NK cells and a decrease of CD56dim NK cells toward the third trimester, indicating a general rather than an MS-specific phenomenon of pregnancy. Additional follow-ups in women with MS showed a reversal of NK cell changes postpartum. Moreover, high-dimensional profiling revealed a specific CD56bright subset with receptor expression related to cytotoxicity and cell activity (e.g., CD16+ NKp46high NKG2Dhigh NKG2Ahigh phenotype) that may drive the expansion of CD56bright NK cells during pregnancy in MS. Our data confirm that pregnancy promotes pronounced shifts of NK cells toward the regulatory CD56bright population. Although exploratory results on in-depth CD56bright phenotype need to be confirmed in larger studies, our findings suggest an increased regulatory NK activity, thereby potentially contributing to disease amelioration of MS during pregnancy.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 106 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.

Background:Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. Methods:The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered withClinicalTrials.gov,NCT01656135,NCT02252055,NCT02085629,NCT02244801,NCT02371434,NCT02129881, andNCT02091232. Findings:The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2–18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. Interpretation:Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression.

The intestine mediates a delicate balance between tolerogenic and inflammatory immune responses. The continuous pathogen encounter might also augment immune cell responses contributing to complications observed upon intestinal transplantation (ITx). We thus hypothesized that ITx patients show persistent signs of immune cell activation affecting both the adaptive and innate immune cell compartment. Information on the impact of intestinal grafts on immune cell composition, however, especially in the long-term is sparse. We here assessed activated and differentiated adaptive and innate immune subsets according to time, previous experience of cellular or antibody-mediated rejections or type of transplant after ITx applying multi-parametric flow cytometry, gene expression, serum cytokine and chemokine profiling. ITx patients showed an increase in CD16 expressing monocytes and myeloid dendritic cells (DCs) compared to healthy controls. This was even detectable in patients who were transplanted more than 10 years ago. Also, conventional CD4+ and CD8+ T cells showed persistent signs of activation counterbalanced by increased activated CCR4+ regulatory T cells. Patients with previous cellular rejections had even higher proportions of CD16+ monocytes and DCs, whereas transplanting higher donor mass with multi-visceral grafts was associated with increased T cell activation. The persistent inflammation and innate immune cell activation might contribute to unsatisfactory results after ITx.

Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLA-B35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF compared to blood regardless of diagnosis.

ObjectiveTo assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model.DesignInvestigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).SettingCharité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).ParticipantsRecipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).InterventionsCD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48.Main outcome measuresThe primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio.ResultsFor all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire.ConclusionsThe application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies.Trial registrationNCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4+ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4+ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development., Despite the current human CD4 memory T cell stratification by CD45RA/CCR7, functional heterogeneities still exist within the respective subsets. Here the authors show that several surface markers, including KLRB1, KLRG1, GPR56 and KLRF1, help to further refine the subsetting of human CD4 memory T cells and provide insights for their differentiation.

The analysis and validation of flow cytometry–based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability 0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman’s correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.

Flow cytometry-based analysis of T-cell receptor (TCR) repertoires is an essential tool for the detection of clonal T-cell expansions in physiologic and pathologic conditions. Individual T-cell subsets can be investigated based on their surface properties. The aims of our study were to provide reference values for various disease settings and delineate the contribution of individual TCR repertoires to the human T-cell differentiation pathway. We analyzed blood of 66 healthy subjects aged 0 (cord blood) to 72 years. Lymphocyte subpopulations and TCR repertoires were simultaneously explored using antibodies specific to CD3, CD4, CD8, CD45RA, CCR7, CD27, CD57 and a set of 25 antibodies detecting human TCR-Vβ chains. Statistical analysis included Wilcoxon, paired t and ANOVA tests. Initially, TCR expansion values were calculated based on the analysis of TCR-Vβ distribution on CD4+ and CD8+ T cells. We then established gating strategies and an algorithm for data analysis allowing for discrimination of T-cell subsets and TCR distribution. Dominant TCR expansions were present within effector as opposed to central/effector memory or naive cells, e.g., median TCR-Vβ expansion rate was highest on CD45RA+/CCR7- effector CD4+/8+ cells (eight and 11-fold, respectively). Remarkably, TCR expansions were missing (0) or very low (0.5) on CD4+ and CD8+ central memory population, respectively. No significant gender-related variability of TCR repertoires was identified, and significant impact of chronic cytomegalovirus infection was shown. Our results serve as reference for future studies elucidating clonal TCR dominance of T-cell subsets.

Purpose of review Transfer of human regulatory T cells (Tregs) has become an attractive therapeutic alternative to improve the long-term outcome in transplantation and thus reduce the side-effects of conventional immunosuppressive drugs. Here, we summarize the recent findings on human Treg subsets, their phenotype and in-vivo function. Recent findings In the last 2 years, it has become apparent that several Treg subsets exist that specifically regulate Th1-driven, Th2-driven, or Th17-driven immune responses; these subsets are very unstable and rapidly change their phenotype, for example, there is loss of Foxp3 expression upon extensive ex-vivo expansion and only the administration of rapamycin has been shown to be able to interfere reproducibly. New humanized mouse models incorporating human solid-organ grafts have been developed, which have been used to test the human Treg in-vivo function, and the first human Treg-cell products have been tested for safety and efficacy in stem cell transplantation. Summary With the recent findings, we have gained a better understanding of Treg heterogeneity, plasticity and function. Using the outcomes of clinical trials in stem cell transplantation, we have learned that adoptive therapy of Tregs is well tolerated and we are now awaiting the first result in solid-organ transplantation from the 'ONE Study'.

Functional characteristics of tuberculosis (TB)-specific CD4 T cells were studied in clinically active pulmonary TB (n = 21) and high TB exposure including LTBI (n = 17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ∼80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-γ accounted for only ∼50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-γ production alone.

Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error-prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age- and gender-dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20-84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The "ONE Study" panels were applied to analyze expression of30 different surface markers to enumerate proportional and absolute numbers of50 leucocyte subsets. Indeed, analysis of the first cohort revealed significant age-dependent changes in subsets e.g. increased activated and differentiated CD4(+) and CD8(+) T cell subsets, acquisition of a memory phenotype for Tregs as well as decreased MDC2 and Marginal Zone B cells. Males and females showed different dynamics in age-dependent T cell activation and differentiation, indicating faster immunosenescence in males. Importantly, although both cohorts consisted of a small sample size, our standardized approach enabled validation of age-dependent changes with the second cohort. Thus, we have proven the utility of our strategy and generated reproducible reference ranges accounting for age- and gender-dependent differences, which are crucial for a better patient monitoring and individualized therapy. © 2016 International Society for Advancement of Cytometry.

Multi-color flow cytometry has become a valuable and highly informative tool for diagnosis and therapeutic monitoring of patients with immune deficiencies or inflammatory disorders. However, the method complexity and error-prone conventional manual data analysis often result in a high variability between different analysts and research laboratories. Here, we provide strategies and guidelines aiming at a more standardized multi-color flow cytometric staining and unsupervised data analysis for whole blood patient samples.

Summary Despite major improvements in short-term survival of organ allografts, long-term graft survival has not changed significantly. It is also known that toxic side effects of current immunosuppressive drugs (IS) especially calcineurin inhibitors (CNI) contribute to the unsatisfactory graft and patient survival following transplantation. Thus, clinicians strive to reduce or wean IS in potentially eligible patients. Research in the last 10 years has focussed on identification of biomarkers suitable for patient stratification in minimization or weaning trials. Most of the described biomarkers have been run retrospectively on samples collected within single-centre trials. Thus, often their performance has not been validated in other potentially multicentre clinical trials. Ultimately, the utility of biomarkers to identify potential weaning candidates should be investigated in large randomized prospective trials. In particular, for testing in such trials, we need more information about the accuracy, reproducibility, stability and limitations of the described biomarkers. Also, data repositories summarizing crucial information on biomarker performance in age- and gender-matched healthy individuals of different ethnicity are missing. This together with improved bioinformatics tools might help in developing better scores for patient stratification. Here, we will summarize the current results, knowledge and limitations on biomarkers for drug minimization or weaning trials.

BACKGROUND: Recent publications have suggested that altered proportions of functional CD4 T-cell subsets correlate with active pulmonary TB. Also, CD27-expression on tuberculin-activated IFN-gamma(+) CD4 T-cells is known to differ significantly between patients with active pulmonary TB and healthy TB-unexposed BCG vaccinees. Here, we explore links between CD4 T-cell phenotype, multiple functional subsets, and control of TB. METHODS: We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-gamma, TNF-alpha, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB. RESULTS: Active pulmonary TB generally showed an excess of TNF-alpha(+) subsets over IFN-gamma(+) subsets, paralleled by decreased CD27 expression on activated IFN-gamma(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-alpha(+) / IFN-gamma(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-alpha(+) /IFN-gamma(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87). CONCLUSIONS: Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-alpha(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease. (c) 2012 International Clinical Cytometry Society.

The application of flow cytometry has hugely advanced the field of tuberculosis (TB) across all areas of research ranging from diagnostic tests to understanding the underlying immunology. As cellular responses are understood to be the mainstay of the immune response in the control of TB it is very likely that polychromatic flow will become the tool of choice for assessing the effects of vaccination. Results are particularly encouraging in this area. The development of a new type of diagnostic test, a prototype of which has been reported, may be the spin-off of a broad and systematic approach to understanding and profiling the T-cell response to TB. It is obvious that flow cytometry will not be able to address all research questions in the field of TB. However, its enormous flexibility as a technology will make it the tool of choice in many situations. An ever increasing availability of flow cytometers, even in resource-poor countries, will rapidly change the face of TB research and management in the years ahead.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub-clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T-cell responses is a promising approach to assessing this situation. For simplicity, class-1 MHC/peptide-multimers staining CD8 T-cells directly are often used but this ignores a much wider range of responses including the whole CD4 T-cell compartment. CD4 T-cells, however, were recently shown to be critical to reducing CMV load early after transplantation. To determine how extensive T-cell responses to CMV are, the responses to two dominant CMV proteins, IE-1 and pp65, were dissected in detail accounting for T-cell lineage, frequencies, epitope recognition and changes over time in more than 25 heart transplant recipients. Cross-sectional results from over 30 healthy CMV-carriers were analyzed for comparison. Responses were unexpectedly complex, with considerable inter-individual variation in terms of dominance, breadth, and recognized epitopes. Whereas the use of MHC/peptide-multimers for clinical CD8 T-cell response monitoring alone can be justified in some situations, short term T-cell activation combined with intracellular cytokine staining was clearly found to be of more general usefulness. The performance of IFN-gamma, TNF-alpha, or IL-2 as single read-outs in identifying activated T-cells was examined and confirmed that the frequently used IFN-gamma was best suited. These results should be used to inform the design of clinically applicable and diagnostically useful approaches to monitoring CMV specific responses in heart transplant recipients.

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. \ud \ud Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. \ud \ud Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.

BACKGROUND: Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells. METHODOLOGY/PRINCIPAL FINDINGS: Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naive/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between. CONCLUSIONS/SIGNIFICANCE: Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

The analysis of T-cell responses to peptides has recently become a busy area of immunologic research. Peptides may be used as single stimulants, pools or libraries, or as part of peptide/major histocompatibility complexes (MHC) for direct T-cell receptor staining. For stimulating T cells, peptides must be bound to MHC molecules. In this study we have used 9- or 10-amino acid peptides, 15-amino acid peptides containing stimulating shorter sequences, and peptides with modified C-terminal function. On average 67% of the T cells from healthy cytomegalovirus-positive donors that bound a frequently used cytomegalovirus pp65/HLA-A*0201 tetramer were able to produce interferon-gamma on stimulation with the respective 9-amino acid peptide. Peptides of 15 amino acids length used at the same concentration (in microg/ml) stimulated CD8 T cells somewhat less efficiently (on average 77% of the frequencies induced with the respective shorter peptides). Modifications of 9-amino acid peptides such as addition of amino acids or functional groups often resulted in a decreased ability to stimulate. However, based on our own results, published data, and theoretic considerations, we conclude that sets of peptides of 15 amino acids length with 11 amino acids overlap represent a good compromise for stimulating both CD8 and CD4 T cells in a number of applications. These parameters may be modified subject to the purpose of a study.

Background No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma‐related muscle injuries using local intraoperative application of allogeneic placenta‐derived, mesenchymal‐like adherent cells (PLX‐PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. Methods Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo‐controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX‐PAD or a placebo (n = 7) into the injured gluteus medius muscles. Results We did not observe any relevant PLX‐PAD‐related adverse events at the 2‐year follow‐up. Improved gluteus medius strength was noted as early as Week 6 in the treatment‐groups. Surprisingly, until Week 26, the low‐dose group outperformed the high‐dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3, 150 M: 237.4 ± 27.2 cm3]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low‐dose treatment reduced the surgery‐related immunological stress reaction more than high‐dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T‐cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late‐onset immune reactivity after high‐dose treatment corresponded to reduced functional improvement. Conclusions Allogeneic PLX‐PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress‐related immunological reactions.