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T cell antigen receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we present a mass cytometric (CyTOF) approach to track T cell responses by combining antibodies for specific TCR Vα and Vβ chains with antibodies against T cell activation and differentiation proteins in mice. This strategy identifies expansions of CD8 + and CD4 + T cells expressing specific Vβ and Vα chains with varying differentiation states in response to Listeria monocytogenes, tumors and respiratory influenza infection. Expanded T cell populations expressing Vβ chains could be directly linked to the recognition of specific antigens from Listeria, tumor cells or influenza. In the setting of influenza infection, we found that common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the TCR diversity and differentiation state of responding T cells. Thus, we present a method to monitor broad changes in TCR use paired with T cell phenotyping during adaptive immune responses., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Treatment of salivary gland tumors (SGTs) remains challenging. Little is known about the immune landscape of SGTs. We aimed to characterize the tumor microenvironment in benign and malignant SGTs., Methods: Eleven benign and nine malignant tumors were collected from patients undergoing curative intent surgery. Specimens were analyzed using mass cytometry by time-of-flight. Immune cell populations were manually gated, and T cells were clustered using the FlowSOM algorithm. Population frequencies were compared between high-grade and low-grade malignancies, corrected for multiple hypothesis testing., Results: There were trends towards increased CD4+ and CD8+ T cells among malignant tumors. High-grade malignancies exhibited trends towards higher frequencies of CD8+ PD-1+ CD39+ CD103+ exhausted T cells, CD4+ FoxP3+ TCF-1+ CD127- Tregs, and CD69+ CD25- CD4+ T cells compared to low-grade malignancies., Conclusion: SGTs exhibit significant immunologic diversity. High-grade malignancies tended to have greater infiltration of exhausted CD8+ T cells and Tregs, which may guide future studies for immunotherapy strategies., (© 2024 The Authors. Head & Neck published by Wiley Periodicals LLC.)

Treatment of penile cancer (PC) focuses on organ preservation, employing various surgical and non-surgical approaches. These interventions may lead to disfigurement, impacting patients' functional outcomes and psychosocial well-being. We reviewed studies related to penile health and PC up to February 2024, limited to studies published in English. Studies employing health-related quality of life (HRQoL) assessments have identified a detrimental association between aggressive treatment and overall health status, physical functioning, and relationships. In contrast, organ-sparing demonstrates improved measures related to HRQoL and sexual function. Assessment through validated questionnaires reveals diverse voiding outcomes, and varying impacts on QoL and sexual activity, emphasizing the necessity for multidisciplinary personalized care. Studies highlight substantial variations in sexual function, with patients reporting adaptations, reduced satisfaction, and concerns about body image and sexual well-being. Furthermore, unmet needs include challenges in patient-clinician communication, obtaining information, and accessing psychosocial support. Patient experiences underscore the importance of timely diagnosis, treatment access, and addressing psychological consequences. Organ-sparing approaches have higher QoL preservation and sexual function. Individualized support, including sexual therapy, support groups, and family counseling, is essential for post-treatment rehabilitation. Timely diagnosis and comprehensive care are paramount in addressing the multifaceted impact of PC on patients and families.

In Alzheimer's disease, tau pathology is thought to spread via a prion-like manner along connected neuronal networks. For this to occur, the usually cytosolic tau protein must be secreted via an unconventional mechanism prior to uptake into the connected neuron. While secretion of healthy and pathological tau has been documented, it remains under-investigated whether this occurs via overlapping or distinct processes. Here, we established a sensitive bioluminescence-based assay to assess mechanisms underlying the secretion of pseudohyperphosphorylated and wild-type tau in cultured murine hippocampal neurons. We found that under basal conditions, both wild-type and mutant tau are secreted, with mutant tau being more robustly secreted. Pharmacological stimulation of neuronal activity led to a modest increase of wild-type and mutant tau secretion, whereas inhibition of activity had no effect. Interestingly, inhibition of heparin sulfate proteoglycan (HSPG) biosynthesis drastically decreased secretion of both wild-type and mutant tau without affecting cell viability. This shows that native and pathological tau share release mechanisms; both activity-dependent and non-activity-dependent secretion of tau is facilitated by HSPGs., Competing Interests: MC was a current employee of Promega UK Ltd. This work was completed before he took up this role. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lopez, Maltby, Warming, Divecha, Vargas-Caballero, Coldwell and Deinhardt.)

CD8 + T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8 + T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8 + T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8 + T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans., Competing Interests: Declaration of interests W.E.O’G. and K.B.J. are employees of Genentech/Roche. M.F.K. is senior advisor and founder of Foundery Immune Studios. M.A. is an inventor on patents related to MIBI technology. M.A. is a consultant, board member, and shareholder in Ionpath Inc. M.H.S. is founder and a board member of Teiko.bio and has received a speaking honorarium from Fluidigm Inc. M.F.K., L.F., A.P.A., P.H., and M.H.S. received research funding from Roche/Genentech., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites., Competing Interests: Declaration of interests R.M.L. is a member of the Scientific Resource Board at Genentech and serves on the Advisory Board at Immunity., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection., Competing Interests: Declaration of interests M.H.S. is a board member and equity holder in Teiko.bio and has received research support from Roche/Genentech, Bristol Myers Squibb, Pfizer, and Valitor. C.S.C. has received funding from NHLBI, FDA, DOD, Genentech, and Quantum Leap Healthcare Collaborative and is on consulting/advisory boards for Vasomune, Gen1e Life Sciences, Janssen, and Cellenkos. C.M.H. has been consulting for Spring Discovery. P.G.W. has a contract from Genentech to study COVID-19., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aimed to propose a simple and efficient heating-freezing method for oil recovery from red tilapia (Oreochromis sp.) viscera, suitable for industrial application and that does not affect its composition. Three methodologies for oil extraction were studied: a) direct heating (69 °C and 29 min) of samples followed by separation of the oil by decantation, b) direct heating with subsequent freezing and c) solvent extraction assisted by ultrasound. For the oil obtained by each methodology, the following factors were determined: peroxide and iodine values, oxidative stability index, yield percentages and fatty acid profile and, to evaluate the changes thereof, a thermal analysis by differential scanning calorimetry was performed. An oil extracted by centrifugation from fresh viscera was used as control. Results showed yields of 92,126%, 60,99% and 55,36% for the oil obtained by heating and freezing, heating and decanting and solvent extraction, respectively, the other evaluated parameters were similar among each other. The content of PUFA was not affected by heating when compared to the control oil, although a decrease was observed in the solvent extracted oil. This behavior was corroborated with the thermal analysis, which showed that the higher PUFA content, the lower the melting temperatures of the oils and the energy required for phase change. A principal component analysis allowed determining that while there are no differences in the abundance of fatty acids C20:1, 14:0, 18:0, 16:1 and C16:0, there are differences for fatty acids C18:1 and C18:2 depending on the method of extraction used in the oil obtention. The results of this study show that the heating-freezing extraction method is a good alternative for acquiring value-added products and facilitates their implementation in rural areas. Furthermore, allows obtaining a product with high content of polyunsaturated fatty acids (at least a third of the total content)., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1 + Ly6C - ) or memory-like status (KLRG1 + Ly6C + ). These pre-existing KLRG1 + Ly6C + NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C - Ly49I - ) NK cells disappeared within both CMV-specific (Ly49H + ) and nonspecific (Ly49H - ) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8 + T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications., Competing Interests: Declaration of interests O.O.O. received honoraria from Kite Pharmaceuticals; consultant fees from Kite, Pfizer, Spectrum, Curio Science, and Bayer; and research funding from Kite. J.C.R. is founder and member of the scientific advisory board of Sitryx Therapeutics; is a member of the scientific advisory boards of Caribou Biosciences and Istesso; and received research funding from Kadmon, Incyte, Tempest, and Calithera and honoraria from Merck and Pfizer. M.H.S. is founder and a board member of Teiko Bio; received consultant fees from Five Prime Therapeutics, Earli, Ono Pharmaceutical, and January; and received research funding from Roche/Genentech, Pfizer, Valitor, and Bristol-Myers Squibb., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor β (TGFβ) is an effector of immune suppression and contributes to a permissive tumor microenvironment that compromises effective immunotherapy. We identified a correlation between TGFB1 and genes expressed by myeloid cells, but not granulocytes, in The Cancer Genome Atlas lung adenocarcinoma data, in which high TGFB1 expression was associated with poor survival. To determine whether TGFβ affected cell fate decisions and lineage commitment, we studied primary cultures of CD14 + monocytes isolated from peripheral blood of healthy donors. We discovered that TGFβ was a survival factor for CD14 + monocytes, which rapidly executed an apoptotic program in its absence. Continued exposure to TGFβ in combination with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL6) amplified HLA-DR low CD14 + CD11b + CD33 + myeloid-derived suppressor cells (MDSCs) at the expense of macrophage and dendritic cell (DC) differentiation. MDSCs generated in the presence of TGFβ were more effective in suppressing T-cell proliferation and promoted the T regulatory cell phenotype. In contrast, inhibition of TGFβ signaling using a small-molecule inhibitor of receptor kinase activity in CD14 + monocytes treated with GM-CSF and IL6 decreased MDSC differentiation and increased differentiation to proinflammatory macrophages and antigen-presenting DCs. The effect of autocrine and paracrine TGFβ on myeloid cell survival and lineage commitment suggests that pharmacologic inhibition of TGFβ-dependent signaling in cancer would favor antitumor immunity., (©2018 American Association for Cancer Research.)

We present a method based on a time-dependent self-consistent density functional tight-binding (TD-DFTB) approach, able to predict the quantum efficiency of the photoinjection process in a dye-TiO 2 complex from a fully atomistic picture. We studied the process of charge transfer of three systems with different dyes: catechol (CAT), alizarin (ALZ) and FSD101. Each system was excited with lasers of different energies in the range of 300-2500 nm, studying the efficiency of the induced charge transfer process at the incident energies. We show that the perturbation can produce either hole transfer or electron transfer from the dye to the nanoparticle, therefore affecting the efficiency of the charge transfer in the solar cell when illuminated by broadband radiation.

The DIII-D Thomson scattering system has been upgraded. A new data acquisition hardware was installed, adding the capacity for additional spatial channels and longer acquisition times for temperature and density measurements. Detector modules were replaced with faster transimpedance circuitry, increasing the signal-to-noise ratio by a factor of 2. This allows for future expansion to the edge system. A second phase upgrade scheduled for 2010-2011 includes the installation of four 1 J/pulse Nd:YAG lasers at 50 Hz repetition rate. This paper presents the first completed phase of the upgrade and performance comparison between the original system and the upgraded system. The plan for the second phase is also presented.