Individual immune cell and cytokine profiles determine platelet-rich plasma composition.

OBJECTIVE: Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. DESIGN: For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. RESULTS: All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. CONCLUSIONS: Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual's immune profile and the concentration method appear to impact the final PRP product. TRIAL REGISTRATION: This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

A Phase I/IIa study of autologous tolerogenic dendritic cells immunotherapy in kidney transplant recipients.

Kidney transplant survival is shortened by chronic rejection and side effects of standard immunosuppressive drugs. Cell-based immunotherapy with tolerogenic dendritic cells has long been recognized as a promising approach to reduce general immunosuppression. Published trials report the safety and the absence of therapy-related adverse reactions in patients treated with tolerogenic dendritic cells suffering from several inflammatory diseases. Here, we present the first phase I clinical trial results using human autologous tolerogenic dendritic cells (ATDC) in kidney transplantation. Eight patients received ATDC the day before transplantation in conjunction with standard steroids, mycophenolate mofetil and tacrolimus immunosuppression with an option to taper mycophenolate mofetil. ATDC preparations were manufactured in a Good Manufacturing Practice-compliant facility and fulfilled cell count, viability, purity and identity criteria for release. A control group of nine patients received the same standard immunosuppression, except basiliximab induction replaced ATDC therapy and mycophenolate tapering was not allowed. During the three-year follow-up, no deaths occurred and there was 100% graft survival. No significant increase of adverse events was associated with ATDC infusion. Episodes of rejection were observed in two patients from the ATDC group and one patient from the control group. However, all rejections were successfully treated by glucocorticoids. Mycophenolate was successfully reduced/stopped in five patients from the ATDC group, allowing tacrolimus monotherapy for two of them. Regarding immune monitoring, reduced CD8 T cell activation markers and increased Foxp3 expression were observed in the ATDC group. Thus, our results demonstrate ATDC administration safety in kidney-transplant recipients.

Widely applicable, extended flow cytometric stem cell enumeration panel for quality control of advanced cellular products.

The most widely used quality control assay for CD34 + hematopoietic stem cell product characterization is the protocol established by the International Society of Hematotherapy and Graft Engineering (ISHAGE). While this protocol is still the gold standard for stem cell enumeration and viability assessment, it does not include T cell enumeration, which is nowadays mandatory for assaying standard allogeneic grafts and various advanced therapy medicinal products (ATMPs). In accordance, we have developed and extensively validated a new approach for a more comprehensive characterization of hematopoietic cellular products using a pre-formulated dried antibody format panel. In addition to the counting beads, the typical markers CD45 fluorescein isothiocyanate (FITC) and CD34 phycoerythrin (PE), as well as the viability dye 7-amino actinomycin D (7-AAD), our novel pre-formulated panel also contains CD3 Pacific Blue (PB) and CD19 allophycocyanin (APC) in the same tube, thereby allowing a combined calculation of leucocytes, stem cells, T and B cells. Showing high linearity, sensitivity and accuracy, our approach is easy to implement and enables a more in-depth characterization of the cellular product under release testing conditions. In addition, the dried pre-formulated antibody approach increases assay reliability compared to the standard antibody panel.

Alterations of NK Cell Phenotype During Pregnancy in Multiple Sclerosis.

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.

Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function.

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.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function.

Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions after transplantation and during autoimmune disease. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. It is therefore critical to scrutinize the molecular changes which occur during T cell product generation, and reexamine current manufacturing practices. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product that has proven safety and hints of therapeutic efficacy in kidney transplant patients. We found progressive DNA methylation changes over the duration of culture, which were donor-independent and reproducible between manufacturing runs. Differentially methylated regions (DMRs) in the final products were significantly enriched at promoters and enhancers of genes implicated in T cell activation. Additionally, significant hypomethylation did also occur in promoters of genes implicated in functional exhaustion in conventional T cells, some of which, however, have been reported to strengthen immunosuppressive effector function in Tregs. At the same time, a set of reported Treg-specific demethylated regions increased methylation levels with culture, indicating a possible destabilization of Treg identity during manufacturing, which was independent of the purity of the starting material. Together, our results indicate that the repetitive TCR-mediated stimulation lead to epigenetic changes that might impact functionality of Treg products in multiple ways, by possibly shifting to an effector Treg phenotype with enhanced functional activity or by risking destabilization of Treg identity and impaired TCR activation. Our analyses also illustrate the value of epigenetic profiling for the evaluation of T cell product manufacturing pipelines, which might open new avenues for the improvement of current adoptive Treg therapies with relevance for conventional effector T cell products.

Dry-fog decontamination of microbiological safety cabinets after activities with SARS-CoV-2: cycle development and process validation for dry fogging with peroxyacetic acid.

Background: Technical protection measures for laboratory activities involving biological agents include biological safety cabinets (BSC) that may be contaminated. In the case of diagnostic activities with SARS-CoV-2, this may also affect BSC that are operated at protection level 2; therefore, decontamination of all contaminated surfaces of the BSC may be required. In addition to fumigation with hydrogen peroxide (H2O2), dry fogging of H2O2-stabilized peroxyacetic acid (PAA) represents another alternative to fumigation with formalin. However, to prove their efficacy, these alternatives need to be validated for each model of BSC. Methods: The validation study was performed on 4 different BSCs of Class II A2 using the "Mini Dry Fog" system. Results: An aerosol concentration of 0.03% PAA and 0.15% H2O2 during a 30 min exposure was sufficient to inactivate SARS-CoV-2. Effective concentrations of 1.0% PAA and 5% H2O2 were required to decontaminate the custom-prepared biological indicators loaded with spores of G. stearothermophilus and deployed at 9 different positions in the BSC. Commercial spore carriers were easier to inactivate by a factor of 4, which corresponded to a reduction of 106 in all localizations. Conclusions: Dry fogging with PAA is an inexpensive, robust, and highly effective decontamination method for BSCs for enveloped viruses such as SARS-CoV-2. The good material compatibility, lack of a requirement for neutralization, low pH - which increases the range of efficacy compared to H2O2 fumigation - the significantly shorter processing time, and the lower costs argue in favor of this method.

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

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.

Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial.

OBJECTIVE: To 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. DESIGN: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS: CD4+ 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 MEASURES: The 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. RESULTS: For 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. CONCLUSIONS: The 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 REGISTRATION: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

Association between Subcutaneous Adipose Tissue Inflammation, Insulin Resistance, and Calorie Restriction in Obese Females.

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.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

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 with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. 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. FUNDING: The 7th EU Framework Programme.

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse. In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements. The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16+ Cell Subsets.

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.

Killer-like receptors and GPR56 progressive expression defines cytokine production of human CD4+ memory T cells.

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, TCRß 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.

Standardized assay for assessment of minimal residual disease in blood, bone marrow and apheresis from patients with plasma cell myeloma.

The recent advances in myeloma treatment result in significantly better outcomes, defined as increased progression free survival (PFS) and overall survival (OS). Since there is a proven correlation between the extend of response and prolonged survival, there is an urgent need for highly sensitive assays for the detection of minimal residual disease (MRD). Next generation flow cytometry has become a valuable approach for sensitive evaluation of the depth of complete response (CR). Here, we report the diagnostic performance and validation results of a single-tube 9-color panel assay. The validation design included intra-assay analysis measuring accuracy, inter-assay analysis estimating method's linearity and precision and inter-assay analysis evaluating repeatability. Furthermore, in inter-operator analysis assessed the comparability of the result analysis of different operators. Staining stability was evaluated in age-of-stain experiments. Our validation results show that a reliable detection of residual myeloma cells is feasible to a detection level of 10-5 with a single-tube assay for a variety of materials (peripheral blood, bone marrow and stem cell apheresis). This study establishes highly sensitive, fully standardized approach for MRD detection in myeloma that is ready for implementation in routine diagnostic laboratories.

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies.

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 <20% and interclass correlation coefficients >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.

Immunomodulatory placental-expanded, mesenchymal stromal cells improve muscle function following hip arthroplasty.

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.

Distinct Housing Conditions Reveal a Major Impact of Adaptive Immunity on the Course of Obesity-Induced Type 2 Diabetes.

Obesity is associated with adipose tissue inflammation, insulin resistance, and the development of type 2 diabetes (T2D). However, our knowledge is mostly based on conventional murine models and promising preclinical studies rarely translated into successful therapies. There is a growing awareness of the limitations of studies in laboratory mice, housed in abnormally hygienic specific pathogen-free (SPF) conditions, as relevant aspects of the human immune system remain unappreciated. Here, we assessed the impact of housing conditions on adaptive immunity and metabolic disease processes during high-fat diet (HFD). We therefore compared diet-induced obesity in SPF mice with those housed in non-SPF, so-called "antigen exposed" (AE) conditions. Surprisingly, AE mice fed a HFD maintained increased insulin levels to compensate for insulin resistance, which was reflected in islet hyperplasia and improved glucose tolerance compared to SPF mice. By contrast, we observed higher proportions of effector/memory T cell subsets in blood and liver of HFD AE mice accompanied by the development of non-alcoholic steatohepatitis-like liver pathology. Thus, our data demonstrate the impact of housing conditions on metabolic alterations. Studies in AE mice, in which physiological microbial exposure was restored, could provide a tool for revealing therapeutic targets for immune-based interventions for T2D patients.