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Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.

People living with HIV (PLWH) are at higher risk of developing lymphoma. In this study, we performed cytometry by time-of-flight (CyTOF) on peripheral blood mononuclear cells of cART-naïve HIV+ individuals and cART-naïve HIV+ individuals prior to AIDS-associated non-Hodgkin lymphoma (pre-NHL) diagnosis. Participants were enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Uniform Manifold Approximation and Projection (UMAP) and unsupervised clustering analysis were performed to identify differences in the expression of B-cell activation markers and/or oncogenic markers associated with lymphomagenesis. CD10+CD27- B cells, CD20+CD27- B cells, and B-cell populations with aberrant features (CD20+CD27+CXCR4+CD71+ B cells and CD20+CXCR4+cMYC+ B cells) were significantly elevated in HIV+ cART-naïve compared to HIV-negative samples. CD20+CD27+CD24+CXCR4+CXCR5+ B cells, CD20+CD27+CD10+CD24+CXCR4+cMYC+ B cells, and a cluster of CD20+CXCR4hiCD27-CD24+CXCR5+CD40+CD4+AICDA+ B cells were significantly elevated in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. A potentially clonal cluster of CD20+CXCR4+CXCR5+cMYC+AICDA+ B cells and a cluster of germinal center B-cell-like cells (CD19-CD20+CXCR4+Bcl-6+PD-L1+cMYC+) were also found in the circulation of HIV+ pre-NHL (cART-naïve) samples. Moreover, significantly elevated clusters of CD19+CD24hiCD38hi cMYC+ AICDA+ B regulatory cells were identified in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. The present study identifies unique B-cell subsets in PLWH with potential pre-malignant features that may contribute to the development of pre-tumor B cells in PLWH and that may play a role in lymphomagenesis.

Background: Infusion of mesenchymal stromal cells (MSCs) has been proposed as immune-modulatory therapy in solid organ transplantation. The use of allogenic MSCs could improve standardization and allow for direct availability of the product. Method: The nonrandomized phase Ib Neptune clinical trial provided safety and feasibility data on the use of allogenic bone-marrow-derived MSCs, infused in 10 patients at week 25 and 26 post kidney transplantation. Here, we performed detailed analysis on the peripheral blood immune cell composition of these patients up to 52 weeks post transplantation. We used a 40 marker antibody panel with mass cytometry to assess potential effects of MSC therapy on the immune system. Results: We showed minor changes in major immune lineages at week 27, 34 and 52 post kidney transplantation after MSC infusion at week 25 and week 26, confirming previous data with regular flow cytometry. However, in a direct comparison between pre- and post MSC infusion, as soon as 4 hours after MSC infusion, we observed a significant increase in cell numbers of B cell and T cell subsets that shared a unique expression of CD11b, CD11c, CD38, CD39, and Ki-67. Conclusion: Exploring these CD11b+CD11c+CD38+CD39+Ki-67+ B cells and T cells in the context of MSC infusion after kidney transplantation may be a promising avenue to better understand the immunological effects of MSC therapy. [ABSTRACT FROM AUTHOR]

Objective: This study aims to comprehensively investigate immune‐cell landscapes in ankylosing spondylitis (AS) patients and explore longitudinal immunophenotyping changes induced by biological agents. Methods: We employed mass cytometry with 35 cellular markers to analyze blood samples from 34 AS patients and 13 healthy controls (HC). Eleven AS patients were re‐evaluated 1 month (4 patients) and 3 months (7 patients) after treatment with biological agents. Flow Self‐Organizing Maps (FlowSOM) clustering was performed to identify specific cellular metaclusters. We compared cellular abundances across distinct subgroups and validated subset differences using gating strategies in flow cytometry scatter plots, visualized with FlowJo software. The proportions of differential subsets were then used for intercellular and clinical correlation analysis, as well as for constructing diagnostic models based on the random forest algorithm. Results: In AS patients, we identified and validated nine different immune‐cell subsets compared to HC. Three subsets increased: helper T‐cell 17 (Th17), mucosa‐associated invariant T‐cell (MAIT), and classical monocytes (CM). Six subsets decreased: effector memory T‐cell (TEM), naïve B cells, transitional B cells, IL10+ memory B cells, non‐classical monocytes (NCM), and neutrophils. Treatments with biological agents could rectify cellular abnormalities, particularly the imbalance of CM/NCM. Furthermore, these subsets may serve as biomarkers for assessing disease activity and constructing effective diagnostic models for AS. Conclusion: These findings provide novel insights into the specific patterns of immune cell in AS, facilitating the further development of novel biomarkers and potential therapeutic targets for AS patients. [ABSTRACT FROM AUTHOR]

Background: Dietary fibers can alter microbial metabolic output in support of healthy immune function; however, the impact of distinct fiber sources and immunomodulatory effects beyond short-chain fatty acid production are underexplored. In an effort to discern the effects of diverse fibers on host immunity, we employed five distinct rodent diets with varying fiber content and source in specific-pathogen-free, gnotobiotic (containing a 14-member synthetic human gut microbiota), and germ-free mice. Results: Broad-scale metabolomics analysis of cecal contents revealed that fiber deprivation consistently reduced the concentrations of microbiota-produced B vitamins. This phenomenon was not always explained by reduced biosynthesis, rather, metatranscriptomic analyses pointed toward increased microbial usage of certain B vitamins under fiber-free conditions, ultimately resulting in a net reduction of host-available B vitamins. Broad immunophenotyping indicated that the local gut effector immune populations and activated T cells accumulate in a microbiota-dependent manner. Supplementation with the prebiotic inulin recovered the availability of microbially produced B vitamins and restored immune homeostasis. Conclusions: Our findings highlight the potential to use defined fiber polysaccharides to boost microbiota-derived B vitamin availability in an animal model and to regulate local innate and adaptive immune populations of the host. 3RFNfpFMTQsk1TPSJTH8Z9 Video abstract. [ABSTRACT FROM AUTHOR]

People living with HIV (PLWH) are at higher risk of developing lymphoma. In this study, we performed cytometry by time-of-flight (CyTOF) on peripheral blood mononuclear cells of cART-naïve HIV+ individuals and cART-naïve HIV+ individuals prior to AIDS-associated non-Hodgkin lymphoma (pre-NHL) diagnosis. Participants were enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Uniform Manifold Approximation and Projection (UMAP) and unsupervised clustering analysis were performed to identify differences in the expression of B-cell activation markers and/or oncogenic markers associated with lymphomagenesis. CD10+CD27- B cells, CD20+CD27- B cells, and B-cell populations with aberrant features (CD20+CD27+CXCR4+CD71+ B cells and CD20+CXCR4+cMYC+ B cells) were significantly elevated in HIV+ cART-naïve compared to HIV-negative samples. CD20+CD27+CD24+CXCR4+CXCR5+ B cells, CD20+CD27+CD10+CD24+CXCR4+cMYC+ B cells, and a cluster of CD20+CXCR4hiCD27-CD24+CXCR5+CD40+CD4+AICDA+ B cells were significantly elevated in HIV+ pre-NHL (cART-naïve) compared to HIV+ cARTnaïve samples. A potentially clonal cluster of CD20+CXCR4+CXCR5+cMYC+AICDA+ B cells and a cluster of germinal center B-cell-like cells (CD19-CD20+CXCR4+Bcl- 6+PD-L1+cMYC+) were also found in the circulation of HIV+ pre-NHL (cART-naïve) samples. Moreover, significantly elevated clusters of CD19+CD24hiCD38hi cMYC+ AICDA+ B regulatory cells were identified in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. The present study identifies unique B-cell subsets in PLWH with potential pre-malignant features that may contribute to the development of pre-tumor B cells in PLWH and that may play a role in lymphomagenesis. [ABSTRACT FROM AUTHOR]

Abstract Clustering is widely used for single-cell analysis, but current methods are limited in accuracy, robustness, ease of use, and interpretability. To address these limitations, we developed an ensemble clustering method that outperforms other methods at hard clustering without the need for hyperparameter tuning. It also performs soft clustering to characterize continuum-like regions and quantify clustering uncertainty, demonstrated here by mapping the connectivity and intermediate transitions between MNIST handwritten digits and between hypothalamic tanycyte subpopulations. This hyperparameter-randomized ensemble approach improves the accuracy, robustness, ease of use, and interpretability of single-cell clustering, and may prove useful in other fields as well.

Abstract Background Dietary fibers can alter microbial metabolic output in support of healthy immune function; however, the impact of distinct fiber sources and immunomodulatory effects beyond short-chain fatty acid production are underexplored. In an effort to discern the effects of diverse fibers on host immunity, we employed five distinct rodent diets with varying fiber content and source in specific-pathogen-free, gnotobiotic (containing a 14-member synthetic human gut microbiota), and germ-free mice. Results Broad-scale metabolomics analysis of cecal contents revealed that fiber deprivation consistently reduced the concentrations of microbiota-produced B vitamins. This phenomenon was not always explained by reduced biosynthesis, rather, metatranscriptomic analyses pointed toward increased microbial usage of certain B vitamins under fiber-free conditions, ultimately resulting in a net reduction of host-available B vitamins. Broad immunophenotyping indicated that the local gut effector immune populations and activated T cells accumulate in a microbiota-dependent manner. Supplementation with the prebiotic inulin recovered the availability of microbially produced B vitamins and restored immune homeostasis. Conclusions Our findings highlight the potential to use defined fiber polysaccharides to boost microbiota-derived B vitamin availability in an animal model and to regulate local innate and adaptive immune populations of the host. Video abstract.

Abstract Background Age-dependent immune responses to coronavirus disease 2019 (COVID-19) vaccinations and breakthrough infections (BIs) in young and middle-aged individuals are unclear. Methods This nationwide multicenter prospective cohort study analyzed immune responses in participants of the ChAdOx1 (ChAd)-ChAd-mRNA vaccine group using cytometry by time-of-flight, anti-spike protein antibody (Sab) and anti-nucleocapsid antibody (Nab) titers, plaque reduction neutralization tests (PRNTs), and interferon-gamma (IFN-γ) release assays at various time points. Results We evaluated 347 participants with an average age of 38.9 ± 9.4 years (range: 21–63). There was a significant inverse correlation between age and Sab levels after the second dose (slope − 14.96, P = 0.032), and this was more pronounced after the third dose (slope − 208.9, P

Moyamoya disease (MMD) is a cerebrovascular disorder marked by progressive arterial narrowing, categorized into six stages known as Suzuki stages based on angiographic features. Growing evidence indicates a pivotal role of systemic immune and inflammatory responses in the initiation and advancement of MMD. This study employs high-dimensional mass cytometry to reveal the immunophenotypic characteristics of peripheral blood immune cells (PBMCs) at various Suzuki stages, offering insights into the progression of MMD. PBMC samples from eight patients with early-stage MMD (Suzuki stages II and III) and eight patients with later-stage MMD (Suzuki stages IV, V, and VI) were analyzed using high-dimensional mass cytometry to evaluate the frequency and phenotype of immune cell subtypes. We identified 15 cell clusters and found that the immunological features of early-stage MMD and later-stage MMD are composed of cluster variations. In this study, we confirmed that, compared to later-stage MMD, the early-stage MMD group exhibits an increase in non-classical monocytes. As the Suzuki stage level increases, the proportions of plasmacytoid DCs and monocyte-derived DCs decrease. Furthermore, T cells, monocytes, DCs, and PMN-MDSCs in the early-stage MMD group show activation of the canonical NF-κB signaling pathway. We summarized and compared the similarities and differences between early-stage MMD patients and later-stage MMD patients. There is a potential role of circulating immune dysfunction and inflammatory responses in the onset and development of MMD. [ABSTRACT FROM AUTHOR]

The pandemic of COVID-19 caused by the SARS-CoV-2 virus is ongoing and a serious menace to global public health. An ocular manifestation is an initial sign of the infection. To date, a comprehensive immune profile of patients with mild COVID-19 has not been well developed. Here, we report a 53-year-old female who noticed a sudden decrease in visual acuity (VA) in both eyes on the fourth day after COVID-19 infection. At presentation (acute phase), the best-corrected VA (BCVA) on the decimal chart was 0.5 in both the right and left eyes. Color fundus photography showed perivascular chorioretinal atrophy with peripheral pigment loss, similar to the fundus appearance of pigmented paravenous chorioretinal atrophy (PPCRA) in the inferior arcade vessels of both eyes. Optical coherence tomography indicated thinning and blurred boundaries of the outer retina in the lesion sites, implying anatomical destruction. She was followed up without any systemic medications. After approximately 15 weeks (remission phase), the BCVA recovered to 0.6 in the right eye and 0.8 in the left. Systemic immune profiles were analyzed using mass cytometry. In the acute phase, monocytes and basophils were dominantly elevated, which suggested the activation of innate immune responses to SARS-CoV-2 and allergic inflammation. In the remission phase, Th2-like cells, plasmablasts, and neutrophils increased predominantly, implying the maturation of adaptive immunity and the preparedness of innate immunity to combat the infection. Our findings indicate that perivascular chorioretinal atrophy resembling PPCRA is a clinical feature of the ocular phenotype of COVID-19, caused by systemic immune responses. [ABSTRACT FROM AUTHOR]

Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI‐67. The characterization of different senescence‐associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single‐cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry‐based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well‐established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy‐related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence‐related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL‐6. Overall, the analysis framework prioritizes multiple senescence‐associated proteins to characterize putative senescent cells sourced from tissue made of different cell types. [ABSTRACT FROM AUTHOR]

Background: Age-dependent immune responses to coronavirus disease 2019 (COVID-19) vaccinations and breakthrough infections (BIs) in young and middle-aged individuals are unclear. Methods: This nationwide multicenter prospective cohort study analyzed immune responses in participants of the ChAdOx1 (ChAd)-ChAd-mRNA vaccine group using cytometry by time-of-flight, anti-spike protein antibody (Sab) and anti-nucleocapsid antibody (Nab) titers, plaque reduction neutralization tests (PRNTs), and interferon-gamma (IFN-γ) release assays at various time points. Results: We evaluated 347 participants with an average age of 38.9 ± 9.4 years (range: 21–63). There was a significant inverse correlation between age and Sab levels after the second dose (slope − 14.96, P = 0.032), and this was more pronounced after the third dose (slope − 208.9, P < 0.001). After BIs, older participants showed significantly higher Sab titers (slope 398.8, P = 0.001), reversing the age-related decline observed post-vaccination. This reversal was also observed in PRNTs against wild-type SARS-CoV-2 and the BA.1 and BA.5 variants. IFN-γ responses increased markedly after the third dose and Bis, but showed a weak positive correlation with age, without statistical significance. Immune cell profiling revealed an age-dependent decrease in the proportions of B-cell lineage cells. The proportions of naive CD4+ and CD8+ T cells were inversely correlated with age, whereas the proportions of mature T cell subsets with memory function, including memory CD4+ T, CD8+ TEM, CD8+ TEMRA, and TFH cells, increased with age. Conclusions: Age-dependent waning of the serologic response to COVID-19 vaccines occurred even in middle-aged individuals, but was reversed after BIs. IFN-γ responses were preserved, compensating for the decrease in naive T cell populations, with an increase in memory T cell populations. [ABSTRACT FROM AUTHOR]

Immune-related skin diseases represent a collective of dermatological disorders intricately linked to dysfunctional immune system processes. These conditions are primarily characterized by an immoderate activation of the immune system or deviant immune responses, involving diverse immune components including immune cells, antibodies, and inflammatory mediators. However, the precise molecular dysregulation underlying numerous individual cases of these diseases and unique subsets respond under disease conditions remains elusive. Comprehending the mechanisms and determinants governing the homeostasis and functionality of diseases could offer potential therapeutic opportunities for intervention. Mass cytometry enables precise and high-throughput quantitative measurement of proteins within individual cells by utilizing antibodies labeled with rare heavy metal isotopes. Imaging mass cytometry employs mass spectrometry to obtain spatial information on cell-to-cell interactions within tissue sections, simultaneously utilizing more than 40 markers. The application of single-cell mass cytometry presents a unique opportunity to conduct highly multiplexed analysis at the single-cell level, thereby revolutionizing our understanding of cell population heterogeneity and hierarchy, cellular states, multiplexed signaling pathways, proteolysis products, and mRNA transcripts specifically in the context of many autoimmune diseases. This information holds the potential to offer novel approaches for the diagnosis, prognostic assessment, and monitoring responses to treatment, thereby enriching our strategies in managing the respective conditions. This review summarizes the present-day utilization of single-cell mass cytometry in studying immune-related skin diseases, highlighting its advantages and limitations. This technique will become increasingly prevalent in conducting extensive investigations into these disorders, ultimately yielding significant contributions to their accurate diagnosis and efficacious therapeutic interventions. [ABSTRACT FROM AUTHOR]

We report the synthesis of a novel class of metal-complexing peptide-based polymers, which we name HyperMAPs (Hyper -loaded M et A l-complexed P olymers). The controlled solid-phase synthesis of HyperMAPs' scaffold peptide provides our polymer with a well-defined molecular structure that allows for an accurate on-design assembly of a wide variety of metals. The peptide-scaffold features a handle for direct conjugation to antibodies or any other biomolecules by means of a thiol-maleimide-click or aldehyde-oxime reaction, a fluorogenic moiety for biomolecule conjugation tracking, and a well-defined number of functional groups for direct incorporation of metal-chelator complexes. Since metal-chelator complexes are prepared in a separate reaction prior to incorporation to the peptide scaffold, polymers can be designed to contain specific ratios of metal isotopes, providing each polymer with a unique CyTOF spectral fingerprint. We demonstrate the complexing of 21 different metals using two different chelators and provide evidence of the application of HyperMAPs on a 13 parameter CyTOF panel and compare its performance to monoisotopic metal-conjugated antibodies. • Current metal chelating polymers for use in mass cytometry design limits amount of metals. • Complexing metals to chelators before attachment to a scaffold provides better quality control. • Our design enables 21 metals and demonstrates similar signal to current polymers. • Spectral deconvolution of multi-isotopic signal is shown, enabling combining isotopes per marker. [ABSTRACT FROM AUTHOR]

Background: Angong Niuhuang Wan (AGNHW, 安宫牛黄丸), is a classical medicinal formula in Traditional Chinese Medicine (TCM) that has been appreciated for its neuroprotective properties in ischemic cerebral injuries, yet its intricate mechanisms remain only partially elucidated. Aims: This study leverages advanced Mass cytometry (CyTOF) to analyze AGNHW's multifaceted immunomodulation effects in‐depth, emphasizing previously underexplored areas. Results: AGNHW mitigated monocyte‐derived macrophages (MoDM) infiltration in the brain, distinguishing its effects on those from microglia. While the vehicle group exhibited elevated inflammatory markers like CD4, CD8a, and CD44 in ischemic brains, the AGNHW‐treated group attenuated their expressions, indicating AGNHW's potential to temper the post‐ischemic inflammatory response. Systemically, AGNHW modulated fundamental immune cell dynamics, notably augmenting CD8+ T cells, B cells, monocytes, and neutrophil counts in the peripheral blood under post‐stroke conditions. Intracellularly, AGNHW exhibited its targeted modulation of the signaling pathways, revealing a remarked inhibition of key markers like IκBα, indicating potential suppression of inflammatory responses in ischemic brain injuries. Conclusion: This study offers a comprehensive portrait of AGNHW's immunomodulation effects on ischemic stroke, illuminating its dual sites of action—both cerebral and systemic—and its nuanced modulation of cellular and molecular dynamics. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5‐year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time‐of‐flight to undertake an in‐depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro‐inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6−CXCR3− memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms. [ABSTRACT FROM AUTHOR]

Objectives: Programmed cell death protein-1 (PD-1) maintains peripheral immune tolerance by preventing T cell continuous activation. Aiming to understand the extent of PD-1 expression in inflammatory arthritis beyond its involvement with T cells, we assess its presence on various circulating single cells. Methods: Mass cytometry analysis of patients with active seropositive/seronegative rheumatoid (RA; n=9/8) and psoriatic (PsA; n=9) arthritis versus healthy controls (HC; n=13), re-evaluating patients after 3 months of anti-rheumatic treatment. Results: PD-1 was expressed in all leukocyte subpopulations, with the highest PD-1+ cell frequencies in eosinophils (59-73%) and T cells (50–60%), and the lowest in natural-killer cells (1–3%). PD-1+ cell frequencies and PD-1 median expression were comparable between patient subgroups and HC, in the majority of cell subsets. Exceptions included increases in certain T cell/B cell subsets of seropositive RA and specific monocyte subsets and dendritic cells of PsA; an expanded PD-1+CD4+CD45RA+CD27+CD28+ T subset, denoting exhausted T cells, was common across patient subgroups. Strikingly, significant inverse correlations between individual biomarkers of systemic inflammation (ESR and/or serum CRP) and PD-1+ cell frequencies and/or median expression were evident in several innate and adaptive immunity cell subsets of RA and PsA patients. Furthermore, all inverse correlations noted in individuals with active arthritis were no longer discernible in those who attained remission/low disease activity post-treatment. Conclusion: PD-1 expression may be insufficient, relative to the magnitude of the concomitant systemic inflammatory response on distinct leukocyte subsets, varying between RA and PsA. Our results point to the potential therapeutic benefits of pharmacological PD-1 activation, to rebalance the autoimmune response and reduce inflammation. [ABSTRACT FROM AUTHOR]

Despite that colorectal and liver cancer are among the most prevalent tumours in the world, the identification of non-invasive biomarkers to aid on their diagnose and subsequent prognosis is a current unmet need that would diminish both their incidence and mortality rates. In this context, conventional flow cytometry has been widely used in the screening of biomarkers with clinical utility in other malignant processes like leukaemia or lymphoma. Therefore, in this review, we will focus on how advanced cytometry panels covering over 40 parameters can be applied on the study of the immune system from patients with colorectal and hepatocellular carcinoma and how that can be used on the search of novel biomarkers to aid or diagnose, prognosis, and even predict clinical response to different treatments. In addition, these multiparametric and unbiased approaches can also provide novel insights into the specific immunopathogenic mechanisms governing these malignant diseases, hence potentially unravelling novel targets to perform immunotherapy or identify novel mechanisms, rendering the development of novel treatments. As a consequence, computational cytometry approaches are an emerging methodology for the early detection and predicting therapies for gastrointestinal cancers. [ABSTRACT FROM AUTHOR]

Vaccination is essential for preventing and controlling infectious diseases, along with reducing mortality. Developing safe and versatile adjuvants to enhance humoral and cellular immune responses to vaccines remains a key challenge in vaccine development. Here, we designed hierarchical mesoporous MOF-801 (HM801) using a Cocamidopropyl betaine (CAPB) and a Pluronics F127 in an aqueous phase system. Meanwhile, we synthesized a novel SARS-CoV-2 nanovaccine (R@M@HM801) with a high loading capacity for both the STING agonist (MSA-2) and the Delta receptor binding domain (Delta-RBD) antigen. R@M@HM801 enhanced MSA-2 and RBD utilization and effectively co-delivered MSA-2 and RBD antigens to antigen-presenting cells in the draining lymph nodes, thereby promoting the activation of both T and B cells. Lymphocyte single-cell analysis showed that R@M@HM801 stimulated robust CD11b+CD4+ T cells, CXCR5+CD4+ T follicular helper (T fh), and durable CD4+CD44+CD62L−, CD8+CD44+CD62L− effector memory T cell (T EM) immune responses, and promoted the proliferative activation of CD26+ B cells in vivo. Meanwhile, R@M@HM801 induced stronger specific antibodies and neutralization of pseudovirus against Delta compared to the RBD + MAS-2 and RBD + MAS-2 + Alum vaccines. Our study demonstrated the efficacy of a hierarchical mesoporous HM801 and its potential immune activation mechanism in enhancing adaptive immune responses against viruses and other diseases. [Display omitted] • HM801 efficiently co-delivers MSA-2 and RBD antigens to DCs in lymph nodes, promotes maturation of DCs, and induces humoral and cellular immunity. • Single-cell analysis indicates a potential synergistic mechanism among DC cells, T cells, and B cells. • R@M@HM801 induces stronger specific antibody responses. • Our study provides new insights into the adjuvants based on hierarchical mesoporous MOFs. [ABSTRACT FROM AUTHOR]

BackgroundInfusion of mesenchymal stromal cells (MSCs) has been proposed as immune-modulatory therapy in solid organ transplantation. The use of allogenic MSCs could improve standardization and allow for direct availability of the product.MethodThe nonrandomized phase Ib Neptune clinical trial provided safety and feasibility data on the use of allogenic bone-marrow-derived MSCs, infused in 10 patients at week 25 and 26 post kidney transplantation. Here, we performed detailed analysis on the peripheral blood immune cell composition of these patients up to 52 weeks post transplantation. We used a 40 marker antibody panel with mass cytometry to assess potential effects of MSC therapy on the immune system.ResultsWe showed minor changes in major immune lineages at week 27, 34 and 52 post kidney transplantation after MSC infusion at week 25 and week 26, confirming previous data with regular flow cytometry. However, in a direct comparison between pre- and post MSC infusion, as soon as 4 hours after MSC infusion, we observed a significant increase in cell numbers of B cell and T cell subsets that shared a unique expression of CD11b, CD11c, CD38, CD39, and Ki-67.ConclusionExploring these CD11b+CD11c+CD38+CD39+Ki-67+ B cells and T cells in the context of MSC infusion after kidney transplantation may be a promising avenue to better understand the immunological effects of MSC therapy.

People living with HIV (PLWH) are at higher risk of developing lymphoma. In this study, we performed cytometry by time-of-flight (CyTOF) on peripheral blood mononuclear cells of cART-naïve HIV+ individuals and cART-naïve HIV+ individuals prior to AIDS-associated non-Hodgkin lymphoma (pre-NHL) diagnosis. Participants were enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Uniform Manifold Approximation and Projection (UMAP) and unsupervised clustering analysis were performed to identify differences in the expression of B-cell activation markers and/or oncogenic markers associated with lymphomagenesis. CD10+CD27- B cells, CD20+CD27- B cells, and B-cell populations with aberrant features (CD20+CD27+CXCR4+CD71+ B cells and CD20+CXCR4+cMYC+ B cells) were significantly elevated in HIV+ cART-naïve compared to HIV-negative samples. CD20+CD27+CD24+CXCR4+CXCR5+ B cells, CD20+CD27+CD10+CD24+CXCR4+cMYC+ B cells, and a cluster of CD20+CXCR4hiCD27-CD24+CXCR5+CD40+CD4+AICDA+ B cells were significantly elevated in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. A potentially clonal cluster of CD20+CXCR4+CXCR5+cMYC+AICDA+ B cells and a cluster of germinal center B-cell-like cells (CD19-CD20+CXCR4+Bcl-6+PD-L1+cMYC+) were also found in the circulation of HIV+ pre-NHL (cART-naïve) samples. Moreover, significantly elevated clusters of CD19+CD24hiCD38hi cMYC+ AICDA+ B regulatory cells were identified in HIV+ pre-NHL (cART-naïve) compared to HIV+ cART-naïve samples. The present study identifies unique B-cell subsets in PLWH with potential pre-malignant features that may contribute to the development of pre-tumor B cells in PLWH and that may play a role in lymphomagenesis.

Alcoholic liver disease (ALD) significantly affects immune cell function and leads to immunological dysregulation. This study explored the potential of granulocyte colony-stimulating factor (G-CSF) to mitigate the negative effects of alcohol on immune cells in a mouse model of ALD. To investigate the capacity of G-CSF, ALD was induced using a 17-day alcohol-enriched diet, followed by a single G-CSF dose prior to sampling. We focused on the dynamics of peripheral blood mononuclear cells using high-dimensional mass cytometry to detect subtle changes. Alcohol intake reduced the number of B cells, monocytes, dendritic cells, and NK cells while increasing the number of T cells. Notably, G-CSF treatment reversed the alcohol-induced increase in total CD4+ and CD8+ T cell populations. This effect was remarkable in naïve, effector CD4+ T cells and naïve CD8+ T cells. PhenoGraph and FlowSOM analysis further revealed the recovery effect of G-CSF on specific T cell subgroups, including central memory CD8+ T cells and double-negative T cells expressing Ly6chighCD44high, which are adversely affected by alcohol. These results enhance our understanding of the effect of ALD on immune function and suggest that G-CSF is a potential therapeutic agent, laying the foundation for future clinical research.

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB-NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment. [ABSTRACT FROM AUTHOR]

Introduction: Systemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets. Methods: Single-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers. Results: Using the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/ CD57– T cells, CD4+/CD57+ T cells, CD8+/CD161– T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4–/CD8– T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3– non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community. [ABSTRACT FROM AUTHOR]

Objective: To construct a molecular immune map of patients with systemic sclerosis (SSc) by mass flow cytometry, and compare the number and molecular expression of double-negative T (DNT) cell subsets between patients and healthy controls (HC). Methods: Peripheral blood mononuclear cells (PBMCs) were extracted from the peripheral blood of 17 SSc patients and 9 HC. A 42-channel panel was set up to perform mass cytometry by time of flight (CyTOF) analysis for DNT subgroups. Flow cytometry was used to validate subpopulation functions. The clinical data of patients were collected for correlation analysis. Results: Compared with HC, the number of total DNT cells decreased in SSc patients. Six DNT subsets were obtained from CyTOF analysis, in which the proportion of cluster1 increased, while the proportion of cluster3 decreased. Further analysis revealed that cluster1 was characterized by high expression of CD28 and CCR7, and cluster3 was characterized by high expression of CD28 and CCR5. After in vitro stimulation, cluster1 secreted more IL-4 and cluster3 secreted more IL-10 in SSc patients compared to HC. Clinical correlation analysis suggested that cluster1 may play a pathogenic role while cluster3 may play a protective role in SSc. ROC curve analysis further revealed that cluster3 may be a potential indicator for determining disease activity in SSc patients. Conclusion: We found a new CCR5+CD28+ DNT cell subset, which played a protective role in the pathogenesis of SSc. Key Points • The number of DNT cells decreased in SSc patients' peripheral blood. • DNT cells do not infiltrate in the skin but secrete cytokines to participate in the pathogenesis of SSc. • A CCR5+CD28+ DNT cell population may play a protective role in SSc. [ABSTRACT FROM AUTHOR]

The maturation of B cells is a complex, multi-step process. During B cell differentiation, errors can occur, leading to the emergence of aberrant versions of B cells that, finally, constitute a malignant tumor. These B cell malignancies are classified into three main groups: leukemias, myelomas, and lymphomas, the latter being the most heterogeneous type. Since their discovery, multiple biological studies have been performed to characterize these diseases, aiming to define their specific features and determine potential biomarkers for diagnosis, stratification, and prognosis. The rise of advanced -omics approaches has significantly contributed to this end. Notably, proteomics strategies appear as promising tools to comprehensively profile the final molecular effector of these cells. In this narrative review, we first introduce the main B cell malignancies together with the most relevant proteomics approaches. Then, we describe the core studies conducted in the field and their main findings and, finally, we evaluate the advantages and drawbacks of flow cytometry, mass cytometry, and mass spectrometry for the profiling of human B cell disorders. [ABSTRACT FROM AUTHOR]

Background: People living with HIV (PLWH) fail to achieve normalization of CD4+ T cell counts and function, especially in immunological non-responders (INRs). The frequencies of Ki67+CD4+ T cells were inversely associated with CD4+ T cell counts in HIV infected patients. Early ART did not normalize CD4+ T cell proliferation. However, the features of the abnormal proliferation CD4+ T cell in INRs are far from known. Method: PLWH were divided into INRs (n= 16) and immunological responders (IRs, n= 53) groups. Mass cytometry was applied to peripheral blood T cells to profile the immune cells and liquid chip technique was used to measure plasma levels of cytokines and chemokines. Correlation analyses were conducted to evaluate associations between the degree of CD4+ T cell proliferation and immune function. Results: The percentage of Ki67+ CD4+ T cells were significant higher in INRs, and we defined these cells with significant higher level of Ki67, as overproliferating cells. No significant difference of markers' expression (HLA-DR, CD38, CD57, PD-1, PD-L1, CD107a, perforin) was found between INRs and IRs. Compared with naïve CD4+ T cells in INRs, Ki67+ CD4+ T cells exhibited lower levels of CD57 and CD38. Whereas Ki67+ T cells exhibited higher levels of CD38 and CD57 and activation compared with differentiated mature central memory CD4+ T cells and effector memory CD4+ T cells. Ki67+ cells did not show higher levels of senescence and activation compared to certain Ki67- CD4+ central memory T cells in IRs. Furthermore, Ki67+ CD4+ Tcm cells exhibited positive correlations with pro-inflammatory cytokines. Conclusion: We proposed and validated the hypothesis of "pathological proliferation" in INRs: excessive proliferation of CD4+ T cells in INRs may be accompanied by aberrant activation, senescence and loss of immune function. Eventually, such over-proliferating but poor-quality cells in INRs result in incomplete recovery of both CD4+ T cell counts and function. An intervention that enhancing the proliferative capacity or functional ability or both of CD4+ T cell in INRs might therefore be beneficial. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of CD45+CD326+ double positive cells (DPCs) on the prognosis of bladder cancer (BLCA), to construct a scoring system for bladder cancer DPCs-related genes, and to explore the characteristics of tumor microenvironment in bladder cancer patients with different DPCs scores. Methods Single-cell sequencing technology was used to analyze the molecular characteristics of DPCs, and mass cytometry was used to analyze the heterogeneity of the tumor microenvironment in patients with high and low proportions of DPCs. Based on the TCGA-BLCA cohort, a scoring system was constructed according to the characteristic genes of DPCs and validated in the GSE13507 cohort and the GSE32894 cohort. Results The prognosis of bladder cancer patients in the high DPCs proportion group was worse than that of patients in the low DPCs proportion group, the tumor microenvironment of patients in the high proportion and low proportion group was heterogeneous, and the immune cells and tumor cells of patients in the low DPCs proportion group had higher expression levels of immune checkpoints, co-inhibitors and coactivators (all P<0.05). A scoring system for bladder cancer with 9 DPCS - related genes was constructed based on TCGA -BLCA cohort (APOBEC3G, CD96, CLEC2D, GNG2, GNLY, IL32, PSMB9, RORA, SKAP1). The results of TCGA-BLCA cohort, GSE13507 cohort and GSE32894 cohort showed that the overall survival of bladder cancer patients in the high DPCs score group was significantly shorter than that of patients in the low DPCs score group (all P<0.01), and the signaling pathways such as immunosuppression and estrogen were significantly enriched in the high-risk score group. Conclusions DPCs are closely related to the poor prognosis of bladder cancer, and the model based on the charactegene of DPCs can accurately predict the clinical prognosis of bladder cancer patients. [ABSTRACT FROM AUTHOR]

ObjectivesProgrammed cell death protein-1 (PD-1) maintains peripheral immune tolerance by preventing T cell continuous activation. Aiming to understand the extent of PD-1 expression in inflammatory arthritis beyond its involvement with T cells, we assess its presence on various circulating single cells.MethodsMass cytometry analysis of patients with active seropositive/seronegative rheumatoid (RA; n=9/8) and psoriatic (PsA; n=9) arthritis versus healthy controls (HC; n=13), re-evaluating patients after 3 months of anti-rheumatic treatment.ResultsPD-1 was expressed in all leukocyte subpopulations, with the highest PD-1+ cell frequencies in eosinophils (59-73%) and T cells (50–60%), and the lowest in natural-killer cells (1–3%). PD-1+ cell frequencies and PD-1 median expression were comparable between patient subgroups and HC, in the majority of cell subsets. Exceptions included increases in certain T cell/B cell subsets of seropositive RA and specific monocyte subsets and dendritic cells of PsA; an expanded PD-1+CD4+CD45RA+CD27+CD28+ T subset, denoting exhausted T cells, was common across patient subgroups. Strikingly, significant inverse correlations between individual biomarkers of systemic inflammation (ESR and/or serum CRP) and PD-1+ cell frequencies and/or median expression were evident in several innate and adaptive immunity cell subsets of RA and PsA patients. Furthermore, all inverse correlations noted in individuals with active arthritis were no longer discernible in those who attained remission/low disease activity post-treatment.ConclusionPD-1 expression may be insufficient, relative to the magnitude of the concomitant systemic inflammatory response on distinct leukocyte subsets, varying between RA and PsA. Our results point to the potential therapeutic benefits of pharmacological PD-1 activation, to rebalance the autoimmune response and reduce inflammation.

Summary: Phenotypic and compositional changes of immune cells in cerebrospinal fluid (CSF) can be used as biomarkers to help diagnose and track disease activity for neuroinflammatory and neurodegenerative diseases. Here, we present a workflow to perform high-dimensional immune profiling at single-cell resolution using cytometry by time-of-flight (CyTOF) on cells isolated from the CSF of patients with neuroinflammation. We describe steps for sample collection and preparation, barcoding to allow for multiplexing, and downstream data analysis using R.For complete details on the use and execution of this protocol, please refer to Fernández-Zapata et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive liver malignancy with limited treatment options and a dismal prognosis. The tumor immune microenvironment (TIME) is crucial for iCCA progression, yet its comprehensive characterization remains incomplete. This study utilized mass cytometry by time of flight (CyTOF) to comprehensively analyze immune cell populations in fresh iCCA tumor samples and adjacent peritumor liver tissues. Notably, NK cell percentages significantly decreased in iCCA lesions compared to peritumor liver tissues. Conversely, an enrichment of immunosuppressive CD39+Foxp3+CD4+ regulatory T cells (CD39+T-regs) and exhausted-like CD8+ T cells (with pronounced CD39 and PD-1 expression) within TIME was identified and confirmed by multiplex immunofluorescence staining in an independent patient cohort (n = 140). Crucially, tumor-infiltrating CD39+T-regs and CD39+PD-1+CD8+ T cells emerged as independent prognostic indicators associated with an unfavorable prognosis in iCCA. These findings unveil the intricate immune landscape within iCCA, offering valuable insights for disease management and novel cancer immunotherapies.

Background: Radiofrequency ablation (RFA) is the primary curative treatment for hepatocellular carcinoma (HCC) patients who are not eligible for surgery. However, the effects of RFA on the global tumor immune response remain unclear. Method: In this study, we examined the phenotypic and functional changes in peripheral blood mononuclear cells (PBMCs) from recurrent HCC patients who had undergone two RFA treatments using mass cytometry and high-throughput mRNA assays. Results: We observed significant increase in monocytes and decrease in T cell subpopulations three days after the first RFA treatment and three days after the second RFA treatment. The down-regulation of GZMB, GZMH, GZMK, and CD8A, which are involved in the cytotoxic function of T cells, was observed following RFA. Furthermore, the population of CD8 effector and memory T cells (CD8 Teff and CD8 Tem) significantly decreased after RFA. The expression of CD5 and CD161 in various T cell subpopulations also showed significant reductions. Additionally, elevated secretion of VEGF was observed in monocytes, B cells, regulatory T cells (Tregs), and CD4 naive T cells. Conclusion: In recurrent HCC patients, serum components derived from radiofrequency therapy can enhance the antigen-presenting capacity of monocytes. However, they also inhibit the anti-cancer immune response by reducing the population of CD8 effector and memory T cells and suppressing the activation of T cells, as well as down-regulating the expression of CD161 and CD5 in various T cell subpopulations. These tumor-derived components also contribute to an immunosuppressive microenvironment by promoting the secretion of VEGF in monocytes, Tregs, B cells, and CD4 naive T cells. [ABSTRACT FROM AUTHOR]

Background: Glioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume. Methods: High-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses. Results: Glioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells. Conclusion: High-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy. [ABSTRACT FROM AUTHOR]

Objectives: Data on peripheral blood mononuclear cells (PBMCs) characteristics of aquaporin‐4 (AQP4)‐IgG seropositive neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody‐associated disease (MOGAD) are lacking. In this study, we describe the whole PBMCs landscape of the above diseases using cytometry by time‐of‐flight mass spectrometry (CyTOF). Methods: The immune cell populations were phenotyped and clustered using CyTOF isolated from 27 AQP4‐IgG seropositive NMOSD, 11 MOGAD patients, and 15 healthy individuals. RNA sequencing was employed to identify critical genes. Fluorescence cytometry and qPCR analysis were applied to further validate the algorithm‐based results that were obtained. Results: We identified an increased population of CD11b+ mononuclear phagocytes (MNPs) in patients with high expression of CCR2, whose abundance may correlate with brain inflammatory infiltration. Using fluorescence cytometry, we confirmed the CCR2+ monocyte subsets in a second cohort of patients. Moreover, there was a wavering of B, CD4+ T, and NKT cells between AQP4‐IgG seropositive NMOSD and MOGAD. Conclusions: Our findings describe the whole landscape of PBMCs in two similar demyelinated diseases and suggest that, besides MNPs, T, NK and B, cells were all involved in the pathogenesis. The identified cell population may be used as a predictor for monitoring disease development or treatment responses. [ABSTRACT FROM AUTHOR]

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients. [ABSTRACT FROM AUTHOR]

Antiviral vaccine is essential for preventing and controlling virus spreading, along with declining morbidity and mortality. A major challenge in effective vaccination lies in the ability to enhance both the humoral and cellular immune responses by adjuvants. Herein, self-assembled nanoparticles based on graphene oxide quantum dots with components of carnosine, resiquimod and Zn2+ ions, namely ZnGC-R, are designed as a new adjuvant for influenza vaccine. With its high capability for antigen-loading, ZnGC-R enhances antigen utilization, improves DC recruitment, and activates antigen-presenting cells. Single cell analysis of lymphocytes after intramuscular vaccination revealed that ZnGC-R generated multifaceted immune responses. ZnGC-R stimulated robust CD4+CCR7loPD-1hi Tfh and durable CD8+CD44hiCD62L- T EM immune responses, and simultaneously promoted the proliferation of CD26+ germinal center B cells. Besides, ZnGC-R elicited 2.53-fold higher hemagglutination-inhibiting antibody than commercial-licensed aluminum salt adjuvant. ZnGC-R based vaccine induced 342% stronger IgG antibody responses compared with vaccines with inactivated virus alone, leading to 100% in vivo protection efficacy against the H1N1 influenza virus challenge. [Display omitted] • ZnGC-R exhibits superior adjuvant activity. • Single cell compositional analysis reveals the potential functional mechanisms among specific DC cells, T cells and B cells. • ZnGC-R induced stronger IgG antibody responses compared with inactivated virus alone, leading to protection efficacy against the H1N1. • Given the long-lasting request of effective adjuvant, our study provides a new insight of self-assembled nanoparticles based on ZnGC-R. [ABSTRACT FROM AUTHOR]

Despite that colorectal and liver cancer are among the most prevalent tumours in the world, the identification of non-invasive biomarkers to aid on their diagnose and subsequent prognosis is a current unmet need that would diminish both their incidence and mortality rates. In this context, conventional flow cytometry has been widely used in the screening of biomarkers with clinical utility in other malignant processes like leukaemia or lymphoma. Therefore, in this review, we will focus on how advanced cytometry panels covering over 40 parameters can be applied on the study of the immune system from patients with colorectal and hepatocellular carcinoma and how that can be used on the search of novel biomarkers to aid or diagnose, prognosis, and even predict clinical response to different treatments. In addition, these multiparametric and unbiased approaches can also provide novel insights into the specific immunopathogenic mechanisms governing these malignant diseases, hence potentially unravelling novel targets to perform immunotherapy or identify novel mechanisms, rendering the development of novel treatments. As a consequence, computational cytometry approaches are an emerging methodology for the early detection and predicting therapies for gastrointestinal cancers.

Background and aimsMetabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition.MethodsMice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning.ResultsIn mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB− NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation.ConclusionOur findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

IntroductionSystemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets.MethodsSingle-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers.ResultsUsing the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/CD57– T cells, CD4+/CD57+ T cells, CD8+/CD161– T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4–/CD8– T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3– non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community.

Anticancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

BackgroundPeople living with HIV (PLWH) fail to achieve normalization of CD4+ T cell counts and function, especially in immunological non-responders (INRs). The frequencies of Ki67+CD4+ T cells were inversely associated with CD4+ T cell counts in HIV infected patients. Early ART did not normalize CD4+ T cell proliferation. However, the features of the abnormal proliferation CD4+ T cell in INRs are far from known.MethodPLWH were divided into INRs (n= 16) and immunological responders (IRs, n= 53) groups. Mass cytometry was applied to peripheral blood T cells to profile the immune cells and liquid chip technique was used to measure plasma levels of cytokines and chemokines. Correlation analyses were conducted to evaluate associations between the degree of CD4+ T cell proliferation and immune function.ResultsThe percentage of Ki67+ CD4+ T cells were significant higher in INRs, and we defined these cells with significant higher level of Ki67, as over-proliferating cells. No significant difference of markers’ expression (HLA-DR, CD38, CD57, PD-1, PD-L1, CD107a, perforin) was found between INRs and IRs. Compared with naïve CD4+ T cells in INRs, Ki67+ CD4+ T cells exhibited lower levels of CD57 and CD38. Whereas Ki67+ T cells exhibited higher levels of CD38 and CD57 and activation compared with differentiated mature central memory CD4+ T cells and effector memory CD4+ T cells. Ki67+ cells did not show higher levels of senescence and activation compared to certain Ki67- CD4+ central memory T cells in IRs. Furthermore, Ki67+ CD4+ Tcm cells exhibited positive correlations with pro-inflammatory cytokines.ConclusionWe proposed and validated the hypothesis of “pathological proliferation” in INRs: excessive proliferation of CD4+ T cells in INRs may be accompanied by aberrant activation, senescence and loss of immune function. Eventually, such over-proliferating but poor-quality cells in INRs result in incomplete recovery of both CD4+ T cell counts and function. An intervention that enhancing the proliferative capacity or functional ability or both of CD4+ T cell in INRs might therefore be beneficial.

The pandemic of COVID-19 caused by the SARS-CoV-2 virus is ongoing and a serious menace to global public health. An ocular manifestation is an initial sign of the infection. To date, a comprehensive immune profile of patients with mild COVID-19 has not been well developed. Here, we report a 53-year-old female who noticed a sudden decrease in visual acuity (VA) in both eyes on the fourth day after COVID-19 infection. At presentation (acute phase), the best-corrected VA (BCVA) on the decimal chart was 0.5 in both the right and left eyes. Color fundus photography showed perivascular chorioretinal atrophy with peripheral pigment loss, similar to the fundus appearance of pigmented paravenous chorioretinal atrophy (PPCRA) in the inferior arcade vessels of both eyes. Optical coherence tomography indicated thinning and blurred boundaries of the outer retina in the lesion sites, implying anatomical destruction. She was followed up without any systemic medications. After approximately 15 weeks (remission phase), the BCVA recovered to 0.6 in the right eye and 0.8 in the left. Systemic immune profiles were analyzed using mass cytometry. In the acute phase, monocytes and basophils were dominantly elevated, which suggested the activation of innate immune responses to SARS-CoV-2 and allergic inflammation. In the remission phase, Th2-like cells, plasmablasts, and neutrophils increased predominantly, implying the maturation of adaptive immunity and the preparedness of innate immunity to combat the infection. Our findings indicate that perivascular chorioretinal atrophy resembling PPCRA is a clinical feature of the ocular phenotype of COVID-19, caused by systemic immune responses.

Summary: Background: Psoriasis is a chronic inflammatory skin disease with a Th17-skewed immune phenotype. Although it has been generally accepted that regulatory T cells (Tregs) in lesional psoriatic skin have functional impairment due to the local inflammatory microenvironment, the molecular properties of skin-homing psoriatic Tregs have not been well explored. Methods: We designed an extensive 39 marker mass cytometry (CyTOF) panel to deeply profile the immune landscape of skin-homing Tregs from 31 people with psoriasis stratified by psoriasis area severity index score as mild (n = 15) to moderate-severe (n = 16) and 32 healthy controls. We further validated the findings with an in-vitro chemokine-mediated Treg migration assay, immunofluorescent imaging of normal and psoriatic lesional skin and analysed public single-cell RNA-sequencing datasets to expand upon our findings into the local tissue microenvironments. Findings: We discovered an overall decrease in CLAhi Tregs and specifically, CLAhiCCR5+ Tregs in psoriasis. Functional markers CD39 and FoxP3 were elevated in psoriatic Tregs. However, CCR7 expression was significantly increased while CCR4 and CLA expression was reduced in psoriatic Tregs and CLAhi Tregs, which was associated with disease severity. Moreover, psoriatic Tregs revealed increased migratory capacity towards CCR7’s ligands, CCL19/CCL21. Interrogation of public single-cell RNA sequencing data confirmed reduced expression of skin-trafficking markers in lesional-skin Tregs compared to non-lesioned skin, further substantiated by immunofluorescent staining. Interpretation: Psoriatic circulating Tregs showed an impaired skin-trafficking phenotype thus leading to insufficient suppression of ongoing inflammation in the lesional skin, expanding upon our current understanding of the impairment of Treg-mediated immunosuppression in psoriasis. Funding: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and Information and Communications Technology (2020R1C1C1014513, 2021R1A4A5032185, 2020R1F1A1073692); and the new faculty research seed money grant of Yonsei University College of Medicine for 2021 (2021-32-0033).

BackgroundGlioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume.MethodsHigh-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses.ResultsGlioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells.ConclusionHigh-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy.

ABSTRACT In the progression of liver disease, various metabolic disorders and inflammatory factors cause dysfunction of intrahepatic leukocytes, but there is no research to summarize the changes in the liver microenvironment driven by different pathological factors, especially HBV. Therefore, this study attempted to explore the inherent laws of disease-driven microenvironment changes in diseased livers. Using mass cytometry, flow cytometry, and immunofluorescence, we analyzed the changes in intrahepatic leukocytes from mouse liver diseases and patients with hepatocellular carcinoma (HCC) in this study. We noticed that (i) the inhibitory effect of hepatitis B virus (HBV) on intrahepatic leukocytes did not increase with the surface antigen titers compared with the low hepatitis B surface antigen concentration group, intrahepatic T cells from the high one showed higher expression of PD-1 and lower expression of TIM-3 and CTLA-4, so the proportion of exhausted T cells decreased in the high one, which contributed to the tolerance or adaptation of the liver to HBV; (ii) leukocytes in HBV-associated HCC were in a highly dysfunctional state with highest PD-1/TIM-3/CTLA-4/TOX; (iii) the detailed leukocyte ratio was appropriate to assess the prognosis of HCC, the ratio of cytotoxic/naive/effector/central memory CD8+ T cells to CD8+ T cells was positively correlated with the overall survival. In conclusion, our phenotype analysis deeply revealed the function differences in intrahepatic leukocytes caused by different liver disease factors, highlighting their unique changes in HBV related HCC, and helping to discover new HCC targeted immunotherapy strategies. IMPORTANCE Hepatitis B virus (HBV)-specific CD8+ T cells play a central role in the clearance of virus and HBV-related liver injury. Acute infection with HBV induces a vigorous, multifunctional CD8+ T cell response, whereas chronic one exhibits a weaker response. Our study elucidated HBV-specific T cell responses in terms of viral abundance rather than the timing of infection. We showed that in the premalignant stage, the degree of impaired T cell function was not synchronized with the viral surface antigen, which was attributed the liver’s tolerance to the virus. However, after the development of hepatocellular carcinoma, T cell exhaustion was inevitable, and it was marked by the exhaustion of the signature transcription factor TOX.

[Display omitted] • A positive role of nasty non-specific binding in biological field was explored. • First universal cell surface barcoding strategy for mass cytometry independent on particular cell surface molecules. • UIO-66 barcode showed high compatibility with commercial MCP mass tags and 16-fold higher sensitivity. • A potential mass/fluorescence dual probe synthetic strategy based on fluorescent dye and PAA/PEG functionalized UIO-66. Non-specific binding was a critical bottleneck in biosensing field and hindered the implementation of biosensors in bioassays. Thus, various approaches were developed to minimize the non-specific binding of biosensors. However, the rational utilization of non-specific binding will also produce generous returns. Herein, the first universal cell surface barcoding strategy for mass cytometry independent on particular cell surface molecules was developed based on nano-sized UIO-66 typed metal organic framework (MOF). Profiting from the 9 abundant stable isotopes of Zr and Hf, up to 84 mass barcode combinations can be achieved theoretically through the non-specific binding between positively charged UIO-66 and negatively charged cells. Clearly distinguished individual population sets were obtained when the cell samples were barcoded with UIO-66(Zr), UIO-66(Hf), and UIO-66(Zr/Hf) mixture. UIO-66 can also be used for flow cytometry barcoding and imaging after the modification with a fluorescent dye. Furthermore, the UIO-66 barcoding showed high compatibility with metal chelated polymer (MCP) mass tags in single-cell multiplexing and 16-fold higher sensitivity than MCP based barcoding agents. This work opens new opportunities for MOFs in mass cytometry applications and will also prompt people to evaluate the nasty non-specific binding in another perspective. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD) significantly affects immune cell function and leads to immunological dysregulation. This study explored the potential of granulocyte colony-stimulating factor (G-CSF) to mitigate the negative effects of alcohol on immune cells in a mouse model of ALD. To investigate the capacity of G-CSF, ALD was induced using a 17-day alcohol-enriched diet, followed by a single G-CSF dose prior to sampling. We focused on the dynamics of peripheral blood mononuclear cells using high-dimensional mass cytometry to detect subtle changes. Alcohol intake reduced the number of B cells, monocytes, dendritic cells, and NK cells while increasing the number of T cells. Notably, G-CSF treatment reversed the alcohol-induced increase in total CD4+ and CD8+ T cell populations. This effect was remarkable in naïve, effector CD4+ T cells and naïve CD8+ T cells. PhenoGraph and FlowSOM analysis further revealed the recovery effect of G-CSF on specific T cell subgroups, including central memory CD8+ T cells and double-negative T cells expressing Ly6chighCD44high, which are adversely affected by alcohol. These results enhance our understanding of the effect of ALD on immune function and suggest that G-CSF is a potential therapeutic agent, laying the foundation for future clinical research. [Display omitted] • We conducted a single-cell-based mass cytometry study to investigate immune cell changes in ALD and G-CSF treated mice. • ALD mice had fewer B cells, monocytes, dendritic cells, and NK cells, but more CD4+ and CD8+ T cells than controls. • G-CSF treatment restored ALD-induced population increases in CD4+ and CD8+ T cells. • This study suggests G-CSF as a potential therapeutic agent for ALD by regulating immune cells and improving liver function. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) constitutes a group of highly persistent man-made substances. Recent evidence indicates that PFAS negatively impact the immune system. However, it remains unclear how different PFAS are associated with alterations in circulating leukocyte subpopulations. More detailed knowledge of such potential associations can provide better understanding into mechanisms of PFAS immunotoxicity in humans. In this exploratory study, associations of serum levels of common PFAS (perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS)) and immune cell profiles of peripheral blood mononuclear cells, both with and without immunostimulation, were investigated. High-dimensional single cell analysis by mass cytometry was done on blood leukocytes from fifty participants in the Norwegian human biomonitoring EuroMix study. Different PFAS were associated with changes in various subpopulations of natural killer (NK), T helper (Th), and cytotoxic T (Tc) cells. Broadly, PFAS concentrations were related to increased frequencies of NK cells and activated subpopulations of NK cells. Additionally, increased levels of activated T helper memory cell subpopulations point to Th2/Th17 and Treg-like skewed profiles. Finally, PFAS concentrations were associated with decreased frequencies of T cytotoxic cell subpopulations with CXCR3+ effector memory (EM) phenotypes. Several of these observations point to biologically plausible mechanisms that may contribute to explaining the epidemiological reports of immunosuppression by PFAS. Our results suggest that PFAS exposures even at relatively low levels are associated with changes in immune cell subpopulations, a finding which should be explored more thoroughly in a larger cohort. Additionally, causal relationships should be confirmed in experimental studies. Overall, this study demonstrates the strength of profiling by mass cytometry in revealing detailed changes in immune cells at a single cell level. • Serum PFAS concentrations were associated with changes in immune cell profiles. • Mass cytometry was done on PBMCs from adults in the EuroMix biomonitoring study. • PFASs were associated with changes in natural killer, T helper, and cytotoxic T cells. • PFASs seem to affect immune cell subpopulations at background exposure levels. [ABSTRACT FROM AUTHOR]