Treg sensitivity to FasL and relative IL-2 deprivation drive idiopathic aplastic anemia immune dysfunction.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell-mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Invadopodia play a role in prostate cancer progression.

BACKGROUND: Invadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation. METHODS: In this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay. RESULTS: Retention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells. CONCLUSION: The combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression.

Costimulation Blockade Disrupts CD4+ T Cell Memory Pathways and Uncouples Their Link to Decline in ß-Cell Function in Type 1 Diabetes.

We previously reported that costimulation blockade by abatacept limits the decline of ß-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of ß-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and ß-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and ß-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in ß-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment.

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

Phenotypic Complexity of the Human Regulatory T Cell Compartment Revealed by Mass Cytometry.

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1ß, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines.

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4-/- knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4-/- cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents.

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4(+)CD25(hi) T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n = 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n = 9). Treg activity, as measured by suppression of proliferation of autologous CD4(+) CD25(-) cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4(+) cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-ß1 (TGF-ß1) levels (P = .002) in patients on treatment, and the levels of TGF-ß1 correlated with the degree of improvement in platelet counts (r = .8, P = .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-ß1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance.

Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.

Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of nonnucleated red cells with their unique discoid shape and membrane material properties. Here we examined the time course of appearance of individual membrane protein components during murine erythropoiesis to throw new light on our understanding of the evolution of the unique features of the red cell membrane. We found that the accumulation of all of the major transmembrane and all skeletal proteins of the mature red blood cell, except actin, accrued progressively during terminal erythroid differentiation. At the same time, and in marked contrast, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and dramatic decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages. These findings provide unique insights into the genesis of red cell membrane function during erythroblast differentiation and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.

An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis.

Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical wealth and objectivity of traditional flow cytometry, overcoming the key limitations of existing approaches for studying asymmetry during mitosis.

Regulatory T-cell status in red cell alloimmunized responder and nonresponder mice.

Red blood cell alloimmunization remains a major complication for transfusion-dependent patients, but immune factors governing risk for alloimmunization are unknown. We hypothesized that CD4+ regulatory T cells (Tregs), which we have shown control the rate and the frequency of red blood cell alloimmunization in mouse models, may dictate responder/nonresponder status. Using a transfusion regimen in which more than 50% of mice develop alloantibodies to human glycophorin A antigen, we found reduced in vitro and in vivo Treg-suppressive activity in responders compared with nonresponders that was the result of impaired Treg suppressor function. Moreover, responders were prone to developing additional alloantibodies to strong immunogens, whereas nonresponders were resistant to alloimmunization. Altogether, our data raise the possibility that Treg activity may be used as a marker for identifying responder/nonresponder status in transfusion recipients.

Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT.

Protein 4.1R (4.1R) was first identified in red cells where it plays an important role in maintaining mechanical stability of red cell membrane. 4.1R has also been shown to be expressed in T cells, but its function has been unclear. In the present study, we use 4.1R-deficient mice to explore the role of 4.1R in T cells. We show that 4.1R is recruited to the immunologic synapse after T cell-antigen receptor (TCR) stimulation. We show further that CD4+ T cells of 4.1R-/- mice are hyperactivated and that they displayed hyperproliferation and increased production of interleukin-2 (IL-2) and interferon gamma (IFNgamma). The hyperactivation results from enhanced phosphorylation of LAT and its downstream signaling molecule ERK. The 4.1R exerts its effect by binding directly to LAT, and thereby inhibiting its phosphorylation by ZAP-70. Moreover, mice deficient in 4.1R display an elevated humoral response to immunization with T cell-dependent antigen. Thus, we have defined a hitherto unrecognized role for 4.1R in negatively regulating T-cell activation by modulating intracellular signal transduction.

Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant.

Human marginal zone B cell development from early T2 progenitors.

B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2, and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of α4ß7 integrin and lower expression of IL-4 receptor (IL4R) compared with the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZBs). Lineage progression from T1 cells to MZBs via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut-homing T2 cells is observed in severe SLE and is associated with reduction of MZBs and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence in health.

A Public Health Antibody Screening Indicates a 6-Fold Higher SARS-CoV-2 Exposure Rate than Reported Cases in Children.

BACKGROUND: Antibody responses to virus reflect exposure and potential protection. METHODS: We developed a highly specific and sensitive approach to measuring antibodies against SARS-CoV-2 for population-scale immune surveillance. Antibody positivity was defined as a dual-positive response against both the receptor-binding domain and nucleocapsid proteins of SARS-CoV-2. Antibodies were measured by immunoprecipitation assays in capillary blood from 15,771 children aged 1 to 18 years living in Bavaria, Germany, and participating in a public health type 1 diabetes screening program (ClinicalTrials.gov: NCT04039945), in 1,916 dried blood spots from neonates in a Bavarian screening study (ClinicalTrials.gov: NCT03316261), and in 75 SARS-CoV-2-positive individuals. Virus positive incidence was obtained from the Bavarian health authority data. FINDINGS: Dual-antibody positivity was detected in none of the 3,887 children in 2019 (100% specificity) and 73 of 75 SARS-CoV-2-positive individuals (97.3% sensitivity). Antibody surveillance in children during 2020 resulted in frequencies of 0.08% in January to March, 0.61% in April, 0.74% in May, 1.13% in June, and 0.91% in July. Antibody prevalence from April 2020 was 6-fold higher than the incidence of authority-reported cases (156 per 100,000 children), showed marked variation between the seven Bavarian regions (p < 0.0001), and was not associated with age or sex. Transmission in children with virus-positive family members was 35%. 47% of positive children were asymptomatic. No association with type 1 diabetes autoimmunity was observed. Antibody frequency in newborns was 0.47%. CONCLUSIONS: We demonstrate the value of population-based screening programs for pandemic monitoring. FUNDING: The work was supported by funding from the BMBF (FKZ01KX1818).

Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is characterized by the presence of antiplatelet autoantibodies as a result of loss of tolerance. CD4+CD25+ regulatory T cells (Tregs) are important for maintenance of peripheral tolerance. Decreased levels of peripheral Tregs in patients with ITP have been reported. To test whether inefficient production or reduced immunosuppressive activity of Tregs contributes to loss of tolerance in patients with chronic ITP, we investigated the frequency and function of their circulating CD4+CD25(hi) Tregs. We found a com-parable frequency of circulating CD4+CD25(hi)Foxp3+ Tregs in patients and controls (n = 16, P > .05). However, sorted CD4+CD25(hi) cells from patients with chronic ITP (n = 13) had a 2-fold reduction of in vitro immunosuppressive activity compared with controls (n = 10, P < .05). The impaired suppression was specific to Tregs as shown by cross-mixing experiments with T cells from controls. These data suggest that functional defects in Tregs contribute to breakdown of self-tolerance in patients with chronic ITP.

Immunophenotyping of Human Peripheral Blood Mononuclear Cells by Mass Cytometry.

Mass cytometry is a variation of conventional flow cytometry using metal tagged antibodies for cell staining instead of fluorochromes and detection in a mass cytometer, a modified mass spectrometer that allows for separation of discrete masses of these metal tags by time of flight (TOF). Currently, up to 50 different metal tags are available for cell analysis. The lack of any significant mass spectral overlap and autofluorescence background makes mass cytometry uniquely suited for complex high-dimensional phenotypic and functional analysis at the single cell level, thus accelerating biomarker discovery and drug screening. Here we describe a workflow for phenotyping of human peripheral blood mononuclear cells (PBMCs) covering cell staining, instrument setup of a Fluidigm Helios™ mass cytometer, and sample acquisition, and summarize a basic workflow of data analysis.

CD41-YFP mice allow in vivo labeling of megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation.

OBJECTIVE: Development of a mouse line permitting live imaging of cells expressing CD41/GpIIb as a means to study megakaryopoiesis. MATERIALS AND METHODS: The gene encoding yellow fluorescent protein (eyfp) was inserted by homologous recombination into embryonic stem cells at the start site of the gpIIb locus. A knockin mouse line, designated CD41-yellow fluorescent protein (YFP), was developed and was characterized by fluorescence microscopy and flow cytometry. Activity of YFP(+) platelets was determined by induction of P-selectin expression in response to thrombin stimulation. RESULTS: CD41-YFP mice contained YFP-labeled megakaryocytes and platelets, the proportions of which varied, depending on the genotype and individual animal, while lymphoid, myelomonocytic, and erythroid lineages were negative. In addition, a fraction of hematopoietic stem cells and intermediate progenitors expressed YFP at low levels. Crossing CD41-YFP mice with lysozyme green fluorescent protein and globin cyan fluorescent protein mice, followed by in vivo imaging of fetal liver, revealed megakaryocytic cells as a subset distinct from myeloid and erythroid cells. This experiment is also the first to show the distribution of three hematopoietic lineages in a minimally perturbed organ. Surprisingly, analysis of CD41-YFP platelets showed that the YFP(+) subset is more responsive to thrombin stimulation than the YFP(-) subset. Experiments aimed at determining the stability of the YFP(+) platelets showed that after lethal irradiation of CD41-YFP mice, the proportion of labeled platelets in the blood declines more rapidly than the bulk of the platelets. CONCLUSION: The newly developed mouse line should become useful not only for in vivo imaging experiments of megakaryocytes and platelets, but also for studies on platelet aging and function. Our irradiation experiments suggest that the YFP(+) platelets are enriched for newly made cells because YFP has a shorter half-life than platelets. Therefore, the finding that YFP(+) platelets are more responsive to thrombin stimulation raises the possibility that platelet activity decreases rapidly during physiological aging.

Autoreactive T effector memory differentiation mirrors ß cell function in type 1 diabetes.

In type 1 diabetes, cytotoxic CD8+ T cells with specificity for ß cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting ß cells. In contrast, the disease relevance of ß cell-reactive CD8+ T cells that are detectable in the circulation, and their relationship to ß cell function, are not known. Here, we tracked multiple, circulating ß cell-reactive CD8+ T cell subsets and measured ß cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in ß cell-specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57- counterparts, and network association modeling indicated that the dynamics of ß cell-reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating ß cell-specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.