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

The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised

CD4(+)CD25(+)FoxP3(+) human regulatory T-CELLS (T-REG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate T-REG to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, T-REG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of T-REG function by suppression of the early activation markers CD154 and CD69 on CD4(+)CD25(-) responder T-CELLS (T-RESP) upon polyclonal stimulation via alpha CD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on T-RESP is due to competition for alpha CD3/28-coated microbeads as opposed to a T-REG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive TREG function

High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges

Background: Immune ageing is a result of repetitive microbial challenges along with cell intrinsic or systemic changes occurring during ageing. Mice under 'specific-pathogen-free' (SPF) conditions are frequently used to assess immune ageing in long-term experiments. However, physiological pathogenic challenges are reduced in SPF mice. The question arises to what extent murine experiments performed under SPF conditions are suited to analyze immune ageing in mice and serve as models for human immune ageing. Our previous comparisons of same aged mice with different microbial exposures, unambiguously identified distinct clusters of immune cells characteristic for numerous previous pathogen encounters in particular in pet shop mice. Results: We here performed single cell mass cytometry assessing splenic as secondary and bone marrow as primary lymphoid organ-derived leukocytes isolated from young versus aged SPF mice in order to delineate alterations of the murine hematopoietic system induced during ageing. We then compared immune clusters from young and aged SPF mice to pet shop mice in order to delineate alterations of the murine hematopoietic system induced by physiological pathogenic challenges and those caused by cell intrinsic or systemic changes during ageing. Notably, distinct immune signatures were similarly altered in both pet shop and aged SPF mice in comparison to young SPF mice, including increased frequencies of memory T lymphocytes, effector-cytokine producing T cells, plasma cells and mature NK cells. However, elevated frequencies of CD4(+) T cells, total NK cells, granulocytes, pDCs, cDCs and decreased frequencies of naive B cells were specifically identified only in pet shop mice. In aged SPF mice specifically the frequencies of splenic IgM(+) plasma cells, CD8(+) T cells and CD4(+) CD25(+) Treg were increased as compared to pet shop mice and young mice. Conclusions: Our study dissects firstly how ageing impacts both innate and adaptive immune cells in primary and secondary lymphoid organs. Secondly, it partly distinguishes murine intrinsic immune ageing alterations from those induced by physiological pathogen challenges highlighting the importance of designing mouse models for their use in preclinical research including vaccines and immunotherapies

Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPβhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68- dendritic cells. Unlike in Crohn's disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS

Virus-associated anterior uveitis and secondary glaucoma: Diagnostics, clinical characteristics, and surgical options

In this retrospective, single-center, observational study, we compared the clinical characteristics, analyzed the glaucoma development, and the glaucoma surgery requirement mediators in patients with different virus-associated anterior uveitis (VAU). In total, 270 patients (= eyes) with VAU confirmed by positive Goldmann-Witmer coefficients (GWC) for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), rubella virus (RV), and multiple virus (MV) were included. Clinical records of these patients were analyzed. Demographic constitution, clinical findings, glaucoma development, and surgeries were recorded. The concentrations of 27 immune mediators were measured in 150 samples of aqueous humor. The GWC analysis demonstrated positive results for CMV in 57 (21%), HSV in 77 (29%), VZV in 45 (17%), RV in 77 (29%), and MV in 14 (5%) patients. CMV and RV AU occurred predominantly in younger and male patients, while VZV and HSV AU appeared mainly with the elderly and females (P<0.0001). The clinical features of all viruses revealed many similarities. In total, 52 patients (19%) showed glaucomatous damage and of these, 27 patients (10%) needed a glaucoma surgery. Minimal-invasive glaucoma surgery (MIGS) showed a reliable IOP reduction in the short-term period. In 10 patients (37%), the first surgical intervention failed and a follow-up surgery was required. We conclude that different virus entities in anterior uveitis present specific risks for the development of glaucoma as well as necessary surgery. MIGS can be suggested as first-line-treatment in individual cases, however, the device needs to be carefully chosen by experienced specialists based on the individual needs of the patient. Filtrating glaucoma surgery can be recommended in VAU as an effective therapy to reduce the IOP over a longer period of time

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

How to Estimate the Probability of Tolerance Long-Term in Liver Transplant Recipients

Background: Operational tolerance as the ability to accept the liver transplant without pharmacological immunosuppression is a common phenomenon in the long-term course. However, it is currently underutilized due to a lack of simple diagnostic support and fear of rejection despite its recognized benefits. In the present work, we present a simple score based on clinical parameters to estimate the probability of tolerance. Patients and methods: In order to estimate the probability of tolerance, clinical parameters from 82 patients after LT who underwent weaning from the IS for various reasons at our transplant center were extracted from a prospectively organized database and analyzed retrospectively. Univariate testing as well as multivariable logistic regression analysis were performed to assess the association of clinical variables with tolerance in the real-world setting. Results: The most important factors associated with tolerance after multivariable logistic regression were IS monotherapy, male sex, history of hepatocellular carcinoma pretransplant, time since LT, and lack of rejection. These five predictors were retained in an approximate model that could be presented as a simple scoring system to estimate the clinical probability of tolerance or IS dispensability with good predictive performance (AUC = 0.89). Conclusion: In parallel with the existence of a tremendous need for further research on tolerance mechanisms, the presented score, after validation in a larger collective preferably in a multicenter setting, could be easily and safely applied in the real world and already now address all three levels of prevention in LT patients over the long-term course

The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised

CD4+CD25+FoxP3+ human regulatory TCELLS (TREG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate TREG to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, TREG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of TREG function by suppression of the early activation markers CD154 and CD69 on CD4+CD25− responder TCELLS (TRESP) upon polyclonal stimulation via αCD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on TRESP is due to competition for αCD3/28-coated microbeads as opposed to a TREG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive TREG function

RESTORE Survey on the Public Perception of Advanced Therapies and ATMPs in Europe — Why the European Union Should Invest More!

Advanced therapy medicinal products (ATMPs) are potential game changers in modern medical care with an anticipated major impact for patients and society. They are a new drug class often referred to as "living drugs," and are based on complex components such as vectors, cells and even tissues. The production of such ATMPs involves innovative biotechnological methods. In this survey, we have assessed the perception of European citizens regarding ATMPs and health care in Europe, in relation to other important topics, such as safety and security, data protection, climate friendly energy supply, migration, and others. A crucial question was to determine to what extent European citizens wish to support public funding of innovations in healthcare and reimbursement strategies for ATMPs. To answer this, we conducted an online survey in 13 European countries (representative of 85.3% of the entire EU population including the UK in 2020), surveying a total of 7,062 European citizens. The survey was representative with respect to adult age groups and gender in each country. Healthcare had the highest ranking among important societal topics. We found that 83% of the surveyed EU citizens were in support of more public funding of technologies in the field of ATMPs. Interestingly, 74% of respondents are in support of cross-border healthcare for patients with rare diseases to receive ATMP treatments and 61% support the reimbursement of very expensive ATMPs within the European health care system despite the current lack of long-term efficacy data. In conclusion, healthcare is a top ranking issue for European Citizens, who additionally support funding of new technologies to enable the wider application of ATMPs in Europe

Differential compartmentalization of myeloid cell phenotypes and responses towards the CNS in Alzheimer's disease

Myeloid cells are suggested as an important player in Alzheimer´s disease (AD). However, its continuum of phenotypic and functional changes across different body compartments and their use as a biomarker in AD remains elusive. Here, we perform multiple state-of-the-art analyses to phenotypically and metabolically characterize immune cells between peripheral blood (n = 117), cerebrospinal fluid (CSF, n = 117), choroid plexus (CP, n = 13) and brain parenchyma (n = 13). We find that CSF cells increase expression of markers involved in inflammation, phagocytosis, and metabolism. Changes in phenotype of myeloid cells from AD patients are more pronounced in CP and brain parenchyma and upon in vitro stimulation, suggesting that AD-myeloid cells are more vulnerable to environmental changes. Our findings underscore the importance of myeloid cells in AD and the detailed characterization across body compartments may serve as a resource for future studies focusing on the assessment of these cells as biomarkers in AD