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To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had >/=2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with 'early' (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and 'intermediate' (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Cytokine flow cytometry (CFC) is a multiparameter assay of antigen-specific T cell function, potentially useful in the monitoring of experimental vaccines and progression of infectious diseases and cancer. Automation of CFC assays would greatly facilitate their use in clinical trials and involves several components. We describe here the migration of these assays to 96-well plates, the use of sample-handling robotics, and the use of lyophilized antigen and antibody plates to help automate CFC. Together, these elements can produce an integrated system capable of walkaway automation of an entire assay, resulting in the reproducible processing of potentially hundreds of samples per day. Implementation of such systems has begun to be undertaken by our group and others. [ABSTRACT FROM AUTHOR]

Summary More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of the data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and larger clinical trials. [ABSTRACT FROM AUTHOR]

MGTA-145 or GROβT, a CXCR2 agonist, has shown promising activity for hematopoietic stem cell (HSC) mobilization with plerixafor in pre-clinical studies and healthy volunteers. Twenty-five patients with multiple myeloma enrolled in a phase 2 trial evaluating MGTA-145 and plerixafor for HSC mobilization (NCT04552743). Plerixafor was given subcutaneously followed 2 h later by MGTA-145 (0.03 mg/kg) intravenously with same day apheresis. Mobilization/apheresis could be repeated for a second day in patients who collected <6 ×10 6 CD34+ cells/kg. Lenalidomide and anti-CD38 antibody were part of induction therapy in 92% (n = 23) and 24% (n = 6) of patients, respectively. Median total HSC cell yield (CD34+ cells/kg × 10 6 ) was 5.0 (range: 1.1-16.2) and day 1 yield was 3.4 (range: 0.3-16.2). 88% (n = 22) of patients met the primary endpoint of collecting 2 ×10 6 CD34+ cells/kg in ≤ two days, 68% (n = 17) in one day. Secondary endpoints of collecting 4 and 6 × 10 6 CD34+ cells/kg in ≤ two days were met in 68% (n = 17) and 40% (n = 10) patients. Grade 1 or 2 adverse events (AE) were seen in 60% of patients, the most common AE being grade 1 pain, usually self-limited. All 19 patients who underwent transplant with MGTA-145 and plerixafor mobilized HSCs engrafted successfully, with durable engraftment at day 100. 74% (17 of 23) of grafts with this regimen were minimal residual disease negative by next generation flow cytometry. Graft composition for HSCs and immune cells were similar to a contemporaneous cohort mobilized with G-CSF and plerixafor., (© 2024. The Author(s).)

A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as 'resisters' (RSTR) show evidence of IFN-γ - T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4 + T cells showed enrichment of T H 17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these T H 17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype., (© 2024. The Author(s).)

Background: Multiple lines of evidence support peripheral organs in the initiation or progression of Lewy body disease (LBD), a spectrum of neurodegenerative diagnoses that include Parkinson's Disease (PD) without or with dementia (PDD) and dementia with Lewy bodies (DLB). However, the potential contribution of the peripheral immune response to LBD remains unclear. This study aims to characterize peripheral immune responses unique to participants with LBD at single-cell resolution to highlight potential biomarkers and increase mechanistic understanding of LBD pathogenesis in humans., Methods: In a case-control study, peripheral mononuclear cell (PBMC) samples from research participants were randomly sampled from multiple sites across the United States. The diagnosis groups comprise healthy controls (HC, n = 159), LBD (n = 110), Alzheimer's disease dementia (ADD, n = 97), other neurodegenerative disease controls (NDC, n = 19), and immune disease controls (IDC, n = 14). PBMCs were activated with three stimulants (LPS, IL-6, and IFNa) or remained at basal state, stained by 13 surface markers and 7 intracellular signal markers, and analyzed by flow cytometry, which generated 1,184 immune features after gating., Results: The model classified LBD from HC with an AUROC of 0.87 ± 0.06 and AUPRC of 0.80 ± 0.06. Without retraining, the same model was able to distinguish LBD from ADD, NDC, and IDC. Model predictions were driven by pPLCγ2, p38, and pSTAT5 signals from specific cell populations under specific activation. The immune responses characteristic for LBD were not associated with other common medical conditions related to the risk of LBD or dementia, such as sleep disorders, hypertension, or diabetes., Conclusions and Relevance: Quantification of PBMC immune response from multisite research participants yielded a unique pattern for LBD compared to HC, multiple related neurodegenerative diseases, and autoimmune diseases thereby highlighting potential biomarkers and mechanisms of disease., (© 2024. The Author(s).)

Introduction: Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing., Methods: To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls., Results: We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC., Discussion: Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection., Competing Interests: The 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kaushik, Chang, Han, He, Komlosi, Ji, Cao, Akdis, Boyd, Pulendran, Maecker, Davis, Chinthrajah, DeKruyff and Nadeau.)

Background: Fludarabine in combination with cyclophosphamide (FC) is the standard lymphodepletion regimen for CAR T-cell therapy (CAR T). A national fludarabine shortage in 2022 necessitated the exploration of alternative regimens with many centers employing single-agent bendamustine as lymphodepletion despite a lack of clinical safety and efficacy data. To fill this gap in the literature, we evaluated the safety, efficacy, and expansion kinetics of bendamustine as lymphodepletion prior to axicabtagene ciloleucel (axi-cel) therapy., Methods: 84 consecutive patients with relapsed or refractory large B-cell lymphoma treated with axi-cel and managed with a uniform toxicity management plan at Stanford University were studied. 27 patients received alternative lymphodepletion with bendamustine while 57 received FC., Results: Best complete response rates were similar (73.7% for FC and 74% for bendamustine, p=0.28) and there was no significant difference in 12-month progression-free survival or overall survival estimates (p=0.17 and p=0.62, respectively). The frequency of high-grade cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome was similar in both the cohorts. Bendamustine cohort experienced lower proportions of hematological toxicities and antibiotic use for neutropenic fever. Immune reconstitution, as measured by quantitative assessment of cellular immunity, was better in bendamustine cohort as compared with FC cohort. CAR T expansion as measured by peak expansion and area under the curve for expansion was comparable between cohorts., Conclusions: Bendamustine is a safe and effective alternative lymphodepletion conditioning for axi-cel with lower early hematological toxicity and favorable immune reconstitution., Competing Interests: Competing interests: DM: Honoraria from Janssen, Fosun Kite Biotechnology; Consulting or Advisory Role with Adaptive Biotechnologies, Juno/Celgene, Pharmacyclics, an AbbVie Company, Janssen. Research Funding from Pharmacyclics, an AbbVie Company, Novartis, Roche/Genentech, Kite, a Gilead Company, Adaptive Biotechnologies, Alimera Sciences, Precision Biosciences, Adicet Bio. LM: consultancy at Pfizer, Amgen, Jazz, Kite, Medexus, CTI Biopharma, and Astellas; research funding from Jasper, Kite, and Astellas; and honoraria from Adaptive. EHM has received research funding 350 from Orca Biosystems. CLM: cofounder of Lyell Immunopharma and Syncopation Life Sciences, which are developing CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar and Immatics. SB reports honoraria (Advisory board) from Allogene. SSyal reports Honoraria (advisory board), Janssen; MJF reports consulting/honoraria from Kite/Gilead, Novartis, BMS, JnJ/Legend, Incyte, and Arcellx. SD has served on scientific advisory board for Kite/Gilead, Bristol Myers Squibb, Incyte Pharma, Adaptive Biotech and has received research funding from Kite/Gilead., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

Age is a major risk factor for severe coronavirus disease 2019 (COVID-19), yet the mechanisms behind this relationship have remained incompletely understood. To address this, we evaluated the impact of aging on host immune response in the blood and the upper airway, as well as the nasal microbiome in a prospective, multicenter cohort of 1031 vaccine-naïve patients hospitalized for COVID-19 between 18 and 96 years old. We performed mass cytometry, serum protein profiling, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays, and blood and nasal transcriptomics. We found that older age correlated with increased SARS-CoV-2 viral abundance upon hospital admission, delayed viral clearance, and increased type I interferon gene expression in both the blood and upper airway. We also observed age-dependent up-regulation of innate immune signaling pathways and down-regulation of adaptive immune signaling pathways. Older adults had lower naïve T and B cell populations and higher monocyte populations. Over time, older adults demonstrated a sustained induction of pro-inflammatory genes and serum chemokines compared with younger individuals, suggesting an age-dependent impairment in inflammation resolution. Transcriptional and protein biomarkers of disease severity differed with age, with the oldest adults exhibiting greater expression of pro-inflammatory genes and proteins in severe disease. Together, our study finds that aging is associated with impaired viral clearance, dysregulated immune signaling, and persistent and potentially pathologic activation of pro-inflammatory genes and proteins.

Introduction: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) presents substantial challenges in patient care due to its intricate multisystem nature, comorbidities, and global prevalence. The heterogeneity among patient populations, coupled with the absence of FDA-approved diagnostics and therapeutics, further complicates research into disease etiology and patient managment. Integrating longitudinal multi-omics data with clinical, health,textual, pharmaceutical, and nutraceutical data offers a promising avenue to address these complexities, aiding in the identification of underlying causes and providing insights into effective therapeutics and diagnostic strategies., Methods: This study focused on an exceptionally severe ME/CFS patient with hypermobility spectrum disorder (HSD) during a period of marginal symptom improvements. Longitudinal cytokine profiling was conducted alongside the collection of extensive multi-modal health data to explore the dynamic nature of symptoms, severity, triggers, and modifying factors. Additionally, an updated severity assessment platform and two applications, ME-CFSTrackerApp and LexiTime, were introduced to facilitate real-time symptom tracking and enhance patient-physician/researcher communication, and evaluate response to medical intervention., Results: Longitudinal cytokine profiling revealed the significance of Th2-type cytokines and highlighted synergistic activities between mast cells and eosinophils, skewing Th1 toward Th2 immune responses in ME/CFS pathogenesis, particularly in cognitive impairment and sensorial intolerance. This suggests a potentially shared underlying mechanism with major ME/CFS comorbidities such as HSD, Mast cell activation syndrome, postural orthostatic tachycardia syndrome (POTS), and small fiber neuropathy. Additionally, the data identified potential roles of BCL6 and TP53 pathways in ME/CFS etiology and emphasized the importance of investigating adverse reactions to medication and supplements and drug interactions in ME/CFS severity and progression., Discussion: Our study advocates for the integration of longitudinal multi-omics with multi-modal health data and artificial intelligence (AI) techniques to better understand ME/CFS and its major comorbidities. These findings highlight the significance of dysregulated Th2-type cytokines in patient stratification and precision medicine strategies. Additionally, our results suggest exploring the use of low-dose drugs with partial agonist activity as a potential avenue for ME/CFS treatment. This comprehensive approach emphasizes the importance of adopting a patient-centered care approach to improve ME/CFS healthcare management, disease severity assessment, and personalized medicine. Overall, these findings contribute to our understanding of ME/CFS and offer avenues for future research and clinical practice., Competing Interests: The 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 © 2024 Jahanbani, Sing, Maynard, Jahanbani, Dafoe, Dafoe, Jones, Wallace, Rastan, Maecker, Röst, Snyder and Davis.)

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4 + T cell and memory B cell responses. Antigen-specific CD4 + T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

The present study aimed at evaluating the YF-specific neutralizing antibody profile besides a multiparametric analysis of phenotypic/functional features of cell-mediated response elicited by the 1/5 fractional dose of 17DD-YF vaccine, administered as a single subcutaneous injection. The immunological parameters of each volunteer was monitored at two time points, referred as: before (Day 0) [Non-Vaccinated, NV (D0) ] and after vaccination (Day 30-45) [Primary Vaccinees, PV (D30-45) ]. Data demonstrated high levels of neutralizing antibodies for PV (D30-45) leading to a seropositivity rate of 93%. A broad increase of systemic soluble mediators with a mixed profile was also observed for PV (D30-45) , with IFN-γ and TNF-α presenting the highest baseline fold changes. Integrative network mapping of soluble mediators showed increased correlation numbers in PV (D30-45) as compared to NV (D0) (532vs398). Moreover, PV (D30-45) exhibited increased levels of Terminal Effector (CD45RA + CCR7 - ) CD4 + and CD8 + T-cells and Non-Classical memory B-cells (IgD + CD27 + ). Dimensionality reduction of Mass Cytometry data further support these findings. A polyfunctional cytokine profile (TNF-α/IFN-γ/IL-10/IL-17/IL-2) of T and B-cells was observed upon in vitro antigen recall. Mapping and kinetics timeline of soluble mediator signatures for PV (D30-45) further confirmed the polyfunctional profile upon long-term in vitro culture, mediated by increased levels of IFN-γ and TNF-α along with decreased production of IL-10. These findings suggest novel insights of correlates of protection elicited by the 1/5 fractional dose of 17DD-YF vaccine., (© 2024. The Author(s).)

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. First, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Second, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Third, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Competing Interests: Competing interests: MTL—Ownership Interest: iRepertoire; Consulting Fees: iRepertoire; KSQ; BioCytics; Zia Pharma; Appel Sauce (pending); Agenus (pending). TC—Consulting Fees: Jannsen. CB—Ownership Interest: PrimeVax; Grant Funding to Institution: Illumina; Consulting Fees: Sanofi, Agilent, Roche, Incendia, PrimeVax, BioAI, Lunaphore. LCh—Consulting Fees: EMD Serono, Jazz Pharmaceuticals, AstraZeneca, Daiichi Sankyo, Ipsen, Nanobiotix, Merck, Sanofi-Genzyme, Regeneron, Bluprint Therapeutics, Beigene, Novartis, Elicio Therapeutics, Alkermes, Gilead, Cullinan-Apollo Corp. SG—Grant Funding to Institution: Regeneron, Boehringer-Ingelheim, Janssen R&D, Genentech, Takeda, Bristol Myers Squibb, Celgene; Patents: Co-inventor on an issued patent for multiplex immunohistochemistry to characterize tumors and treatment responses. The technology is filed through Icahn School of Medicine at Mount Sinai (ISMMS) and is currently unlicensed, Inventor of technology to detect autoantibodies to GM-CSF, a cytokine that promotes myeloid cell development and maturation. The technology is filed through the Icahn School of Medicine at Mount Sinai (ISMMS) and is currently unlicensed, Inventor on a patent application NY-ESO-1 Polypeptides filed through Mount Sinai and Ludwig Institute. This technology is currently unlicensed. HTM—Consulting Fees: Caris Life Sciences, Cytek Biosciences; Stock Holdings: Caris Life Sciences, Cytek Biosciences. LCo and JYPS—Nothing to disclose. SITC Staff (PJI and ER)—Nothing to disclose.

Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.

The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID-19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease., (© 2024. The Author(s).)

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC., (© 2024. The Author(s).)

Multiparameter flow cytometry is a common tool for assessing responses of T, B, and other cells to pathogens or vaccines. Such responses are likely to be important for predicting the efficacy of an HIV vaccine, despite the elusive findings in HIV vaccine trials to date. Fortunately, flow cytometry has evolved to be capable of readily measuring 30-40 parameters, providing the ability to dissect detailed phenotypes and functions that may be correlated with disease protection. Nevertheless, technical hurdles remain, and standardization of assays is still largely lacking. Here an optimized protocol for antigen-specific T cell monitoring is presented, with specific variations for particular markers. It covers the analysis of multiple cytokines, cell surface proteins, and other functional markers such as CD107, CD154, CD137, etc. References are given to published panels of 8-28 colors., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

T cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, CyTOF ® mass cytometry is used to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. A method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69, is also included. Coupling magnetic enrichment with highly multiparameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The clinical trajectory of COVID-19 may be influenced by previous responses to heterologous viruses. We examined the relationship of Abs against different viruses to clinical trajectory groups from the National Institutes of Health IMPACC (Immunophenotyping Assessment in a COVID-19 Cohort) study of hospitalized COVID-19 patients. Whereas initial Ab titers to SARS-CoV-2 tended to be higher with increasing severity (excluding fatal disease), those to seasonal coronaviruses trended in the opposite direction. Initial Ab titers to influenza and parainfluenza viruses also tended to be lower with increasing severity. However, no significant relationship was observed for Abs to other viruses, including measles, CMV, EBV, and respiratory syncytial virus. We hypothesize that some individuals may produce lower or less durable Ab responses to respiratory viruses generally (reflected in lower baseline titers in our study), and that this may carry over into poorer outcomes for COVID-19 (despite high initial SARS-CoV-2 titers). We further looked at longitudinal changes in Ab responses to heterologous viruses, but found little change during the course of acute COVID-19 infection. We saw significant trends with age for Ab levels to many of these viruses, but no difference in longitudinal SARS-CoV-2 titers for those with high versus low seasonal coronavirus titers. We detected no difference in longitudinal SARS-CoV-2 titers for CMV seropositive versus seronegative patients, although there was an overrepresentation of CMV seropositives among the IMPACC cohort, compared with expected frequencies in the United States population. Our results both reinforce findings from other studies and suggest (to our knowledge) new relationships between the response to SARS-CoV-2 and Abs to heterologous viruses., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The dynamics of immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infants and young children by analyzing blood samples and weekly nasal swabs collected before, during, and after infection with Omicron and non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, showed no sign of decay for up to 300 days. Infants mounted a robust mucosal immune response characterized by inflammatory cytokines, interferon (IFN) α, and T helper (Th) 17 and neutrophil markers (interleukin [IL]-17, IL-8, and CXCL1). The immune response in blood was characterized by upregulation of activation markers on innate cells, no inflammatory cytokines, but several chemokines and IFNα. The latter correlated with viral load and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell multi-omics. Together, these data provide a snapshot of immunity to infection during the initial weeks and months of life., Competing Interests: Declaration of interests B.P. serves on the External Immunology Board of GSK and on the Scientific Advisory Board of Sanofi, Medicago, Boehringer Ingelheim, Icosavax, and EdJen. F.W. is a consultant for Gilead. A.S. is a consultant for Gritstone Bio, Flow Pharma, Moderna, AstraZeneca, Qiagen, Fortress, Gilead, Sanofi, Merck, RiverVest, MedaCorp, Turnstone, NA Vaccine Institute, Emervax, Gerson Lehrman Group and Guggenheim. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: We compare cytokine profiles at the time of initial CSF shunt placement between children who required no subsequent shunt revision surgeries and children requiring repeated CSF shunt revision surgeries for CSF shunt failure. We also describe the cytokine profiles across surgical episodes for children who undergo multiple subsequent revision surgeries., Methods: This pilot study was nested within an ongoing prospective multicenter study collecting CSF samples and clinical data at the time of CSF shunt surgeries since August 2014. We selected cases where CSF was available for children who underwent an initial CSF shunt placement and had no subsequent shunt revision surgeries during >=24 months of follow-up (n = 7); as well as children who underwent an initial CSF shunt placement and then required repeated CSF shunt revision surgeries (n = 3). Levels of 92 human cytokines were measured using the Olink immunoassay and 41 human cytokines were measured using Luminex based bead array on CSF obtained at the time of each child's initial CSF shunt placement and were displayed in heat maps., Results: Qualitatively similar profiles for the majority of cytokines were observed among the patients in each group in both Olink and Luminex assays. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Pro- and anti-inflammatory cytokines were selected a priori and shown across subsequent revision surgeries for the 3 patients. Cytokine patterns differed between patients, but within a given patient pro-inflammatory and anti-inflammatory cytokines acted in a parallel fashion, with the exception of IL-4., Conclusions: Heat maps of cytokine levels at the time of initial CSF shunt placement for each child undergoing only a single initial CSF shunt placement and for each child undergoing repeat CSF shunt revision surgeries demonstrated qualitatively similar profiles for the majority of cytokines. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Better stratification by patient age, etiology, and mechanism of failure is needed to develop a deeper understanding of the mechanism of inflammation in the development of hydrocephalus and response to shunting in children., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tamara Simon reports financial support was provided by National Institutes of Health. Tamara Simon reports financial support was provided by National Center for Advancing Translational Sciences. David D. Limbrick reports a relationship with Mircobot Medical Inc. that includes: funding grants. No other conflict of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals., (Copyright © 2023 The Authors.)

Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved-in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two-pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our 'EVROS' strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 µl sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion., (© 2023. The Author(s).)

Background: Multiplexed protein analysis platforms are a novel and efficient way to characterize biomarkers in a variety of biological samples. Few studies have compared protein quantitation and reproducibility of results across platforms. We utilize a novel nasosorption technique to collect nasal epithelial lining fluid (NELF) from healthy subjects, and compare the detection of proteins in NELF across three commonly used platforms., Methods: NELF was collected from both nares of twenty healthy subjects using an absorbent fibrous matrix and analyzed using three different protein analysis platforms: Luminex, Meso Scale Discovery (MSD), and Olink. Twenty-three protein analytes were shared across two or more platforms, and correlations across platforms were assessed using Spearman correlations., Results: Among the twelve proteins represented on all three platforms, IL1⍺ and IL6 were very highly correlated (Spearman correlation coefficient [r] ≥ 0.9); CCL3, CCL4, and MCP1 were highly correlated (r ≥ 0.7); and IFNɣ, IL8, and TNF⍺ were moderately correlated (r ≥ 0.5). Four proteins (IL2, IL4, IL10, IL13) were poorly correlated across at least two platform comparisons (r < 0.5); for two of these proteins (IL10 and IL13), the majority of observations were below the limits of detection for Olink and Luminex., Discussion: Multiplexed protein analysis platforms are a promising method for analyzing nasal samples for biomarkers of interest in respiratory health research. For most proteins evaluated, there was good correlation across platforms, although results were less consistent for low abundance proteins. Of the three platforms tested, MSD had the highest sensitivity for analyte detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with regard to the content of this report., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Type I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown., Methods: Using high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL., Results: We find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYC high human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15., Conclusion: We find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL., Competing Interests: Competing interests: The work described in this study is covered by pending US and PCT patent applications assigned to City of Hope and/or Stanford University with inventors SS, AK, ATK, CW-C, SJL, CD and HTM. MAC and JY are cofounders of CytoImmune Therapeutics., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: Cytokine profiles of peripheral blood and other bodily fluids provide diagnostic indicators for assessing inflammatory processes. Menstrual effluent may provide a noninvasive source of biological material for monitoring cytokine levels in blood and in endometrial tissues. This pilot study investigated the potential of measuring cytokines in menstrual effluent, and compared the cytokine profiles of menstrual versus peripheral blood., Methods: Seven healthy donors (aged ≥18 and ≤45 years) collected menstrual effluent on day 2 of menses. Matched peripheral blood samples were collected by venous blood draw on the same day. Levels of 62 cytokines were measured in all samples by 62-plex Luminex assay., Results: Peripheral blood and menstrual effluent cytokine profiles were tenuously correlated ( r 2  = 0.26, p  < 0.0001), with higher levels detected in menstrual effluent for 48/62 cytokines. Thirty five cytokines were significantly elevated in menstrual effluent compared to peripheral blood samples (IL-8, CCL2, CCL4, LIF, IL-1RA, IL-6, IL-1β, HGF, CCL3, FGF-2, TNF-α, VEGF-A, IL-1α, CXCL1, IL-9, IL-10, EGF, CXCL5, CSF3, EOTAXIN, TGF-α, TRAIL, CXCL10, VEGF-D, IL-12P40, CXCL9, IL-18 RESISTIN, IL-22, IL-21, CSF1, IFN-γ, IL-17A, CXCL12, IL-12p70). Two cytokines (LEPTIN, CSF2) were expressed at significantly lower levels in menstrual effluent compared to peripheral blood. Linear regression of individual cytokines found low predictive power (linear regression p  > 0.05) for 53/62 cytokines in menstrual effluent versus peripheral blood. Levels of TGF-β ( r 2  = 0.87, p  = 0.002) and CCL7 ( r 2  = 0.63, p  = 0.033) were significantly positively correlated between matched menstrual and peripheral blood samples., Conclusion: In this group of study participants, the cytokine profile of menstrual effluent was quantitatively distinct from peripheral blood, and also characterized by higher levels of inflammatory signaling. This pattern of comparative menstrual blood cytokine profiles points to a need for further studies to evaluate the relationship between peripheral and menstrual blood cytokines in broader populations including both healthy and diseased states., Competing Interests: S. Naseri and M. Avrutsky are employed by Qurasense Inc., which develops laboratory tests for analyzing menstrual blood. Funding sources and financial relationships had no role in study design, collection, analysis, or interpretation of data., (© 2023 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Competing Interests: This work was supported by National Institutes of Health grant 1K08HL16140501; a Sarnoff Scholar Career Development Award; National Institute of General Medical Sciences grant 1RM1 GM131981-02; an American Heart Association Established Investigator Award; the Hoffmann/Schroepfer Foundation; the Additional Venture Foundation; the Joan and Sanford I. Weill Scholar Fund; an iAward grant from Sanofi US; National Heart, Lung, and Blood Institute grant R01HL13483004; and American Heart Association grant AW849785 (Transformative Award). Dr Wu has received funding from Sanofi. Dr Witteles sits on advisory boards for Pfizer, Alnylam, Ionis/Akcea, Eidos, and Intelia. Dr Neal has served in a consulting or an advisory role for AstraZeneca, Genentech/Roche, Exelixis, Jounce Therapeutics, Takeda Pharmaceuticals, Eli Lilly, Calithera Biosciences, Amgen, Iovance, Biotherapeutics, Blueprint Pharmaceuticals, Regeneron Pharmaceuticals, and Natera; and has received research funding from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Nektar Therapeutics, Takeda Pharmaceuticals, Adaptimmune, GlaxoSmithKline, Janssen, and AbbVie. Dr Waliany has received consulting fees from AstraZeneca. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

While food allergy oral immunotherapy (OIT) can provide safe and effective desensitization (DS), the immune mechanisms underlying development of sustained unresponsiveness (SU) following a period of avoidance are largely unknown. Here, we compare high dimensional phenotypes of innate and adaptive immune cell subsets of participants in a previously reported, phase 2 randomized, controlled, peanut OIT trial who achieved SU vs. DS (no vs. with allergic reactions upon food challenge after a withdrawal period; n = 21 vs. 30 respectively among total 120 intent-to-treat participants). Lower frequencies of naïve CD8 + T cells and terminally differentiated CD57 + CD8 + T cell subsets at baseline (pre-OIT) are associated with SU. Frequency of naïve CD8 + T cells shows a significant positive correlation with peanut-specific and Ara h 2-specific IgE levels at baseline. Higher frequencies of IL-4 + and IFNγ + CD4 + T cells post-OIT are negatively correlated with SU. Our findings provide evidence that an immune signature consisting of certain CD8 + T cell subset frequencies is potentially predictive of SU following OIT., (© 2022. The Author(s).)

Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Background: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune-related adverse events such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T cell-mediated mechanisms. However, the precise pathological immune subsets and molecular changes in ICI myocarditis are unknown., Methods: To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry on peripheral blood mononuclear cells from 52 individuals: 29 patients with autoimmune adverse events (immune-related adverse events) on ICI, including 8 patients with ICI myocarditis, and 23 healthy control subjects. We also used multiomics single-cell technology to immunophenotype 30 patients/control subjects using single-cell RNA sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes by sequencing with feature barcoding for surface marker expression confirmation. To correlate between the blood and the heart, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing on MRL/Pdcd1 -/- (Murphy Roths large/programmed death-1-deficient) mice with spontaneous myocarditis., Results: Using these complementary approaches, we found an expansion of cytotoxic CD8 + T effector cells re-expressing CD45RA (Temra CD8 + cells) in patients with ICI myocarditis compared with control subjects. T-cell receptor sequencing demonstrated that these CD8 + Temra cells were clonally expanded in patients with myocarditis compared with control subjects. Transcriptomic analysis of these Temra CD8 + clones confirmed a highly activated and cytotoxic phenotype. Longitudinal study demonstrated progression of these Temra CD8 + cells into an exhausted phenotype 2 months after treatment with glucocorticoids. Differential expression analysis demonstrated elevated expression levels of proinflammatory chemokines (CCL5/CCL4/CCL4L2) in the clonally expanded Temra CD8 + cells, and ligand receptor analysis demonstrated their interactions with innate immune cells, including monocytes/macrophages, dendritic cells, and neutrophils, as well as the absence of key anti-inflammatory signals. To complement the human study, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing in Pdcd1 -/- mice with spontaneous myocarditis and found analogous expansions of cytotoxic clonal effector CD8 + cells in both blood and hearts of such mice compared with controls., Conclusions: Clonal cytotoxic Temra CD8 + cells are significantly increased in the blood of patients with ICI myocarditis, corresponding to an analogous increase in effector cytotoxic CD8 + cells in the blood/hearts of Pdcd1 -/- mice with myocarditis. These expanded effector CD8 + cells have unique transcriptional changes, including upregulation of chemokines CCL5/CCL4/CCL4L2, which may serve as attractive diagnostic/therapeutic targets for reducing life-threatening cardiac immune-related adverse events in ICI-treated patients with cancer.

Background: People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively., Aim: To establish whether saffron treatment alleviates inflammation in experimental colitis., Methods: Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25 mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis., Results: Saffron 20 mg/kg body weight showed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10 + dendritic cells. Saffron treatment also enhanced CD3 + T and CD3 + CD8 + T cells followed by increase in different CD3 + CD4 + T cells subsets like CD25 + T cells, FoxP3 + CD25 + regulatory T cells, and CD4 + FOXP3 + CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways., Conclusion: These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Improving our understanding of the immunologic response to cancer cells within the sentinel lymph nodes (SLN) of primary tumors is expected to identify new approaches to stimulate clinically meaningful cancer immunity., Experimental Design: We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T-cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of patients with melanoma., Results: We found increased effector-memory αβ T cells, TCR clonality, and γδ T cells selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an antitumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells, which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared with non-melanoma-bearing SLNs include (i) reduced CD8+CD69+ T cell/T regulatory cell ratio, (ii) high PD-1 expression on CD4+ and CD8+ T cells, and (iii) high CTLA-4 expression on γδ T cells., Conclusions: Our data suggest that these immunologic changes compromise antimelanoma immunity and contribute to a high relapse rate. We propose the development of clinical trials to test the neo-adjuvant administration of anti-PD-1 antibodies prior to SLN resection in patients with stage III melanoma. See related commentary by Lund, p. 1996., (©2022 American Association for Cancer Research.)

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.

Adolescent depression is characterized by heightened inflammation and altered connectivity of fronto-cingulate-limbic tracts, including the genu of the corpus callosum (CCG) and the uncinate fasciculus (UF). No studies, however, have yet examined the association between inflammation, measured by peripheral levels of cytokines, and white matter connectivity of fronto-cingulate-limbic tracts in adolescents. Here, 56 depressed adolescents (32 females, 3 non-binary; 16.23 ± 1.28 years) and 19 controls (10 females; 15.72 ± 1.17 years) completed a diffusion-weighted MRI scan at 3 Tesla. We conducted deterministic tractography to segment bilateral corpus callosum (genu and splenium) and UF and computed mean fractional anisotropy (FA) in each tract. A subset of participants (43 depressed and 17 healthy controls) also provided dried blood spot samples from which we assayed interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-ɑ) using a Luminex multiplex array. Depressed participants did not differ from controls in FA of the corpus callosum or UF (all FDR-corrected ps > 0.056) but exhibited higher levels of inflammation than did controls (IL-6: β = 0.91, FDR-corrected p = 0.006; TNF-α: β = 0.76, FDR-corrected p = 0.006). Although diagnostic group did not moderate the associations between inflammatory cytokines and FA in the CCG and UF, across both groups, greater peripheral inflammation was associated with lower FA in the CCG (IL-6: β = -0.38; FDR-corrected p = 0.044; TNF-ɑ: β = -0.41, FDR-corrected p = 0.044). This study is the first to examine associations between peripheral inflammation and white matter microstructure of fronto-cingulate-limbic tracts in depressed and nondepressed adolescents. Future mechanistic studies are needed to confirm our findings; nevertheless, our results suggest that heightened inflammation is an important component of neurophenotypes that are relevant to adolescent depression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The retention of the heavy metal, gadolinium, after a Gadolinium-Based Contrast Agent-assisted MRI may lead to a symptom cluster termed Gadolinium Deposition Disease. Little is known of the disorder's underlying pathophysiology, but a recent study reported abnormally elevated serum levels of pro-inflammatory cytokines compared to normal controls. As a calcium channel blocker in cellular plasma and mitochondrial membranes, gadolinium also interferes with mitochondrial function. We applied to sera from nine Gadolinium Deposition Disease and two Gadolinium Storage Condition patients newly developed methods allowing isolation of plasma neuron-derived extracellular vesicles that contain reproducibly quantifiable levels of mitochondrial proteins of all major classes. Patients' levels of five mitochondrial functional proteins were statistically significantly lower and of two significantly higher than the levels in normal controls. The patterns of differences between study patients and controls for mitochondrial dynamics and mitochondrial proteins encompassing neuronal energy generation, metabolic regulation, ion fluxes, and survival differed from those seen for patients with first episode psychosis and those with Major Depressive Disorder compared to their controls. These findings suggest that mitochondrial dysfunction due to retained gadolinium may play a role in causing Gadolinium Deposition Disease. Larger samples of both GDD and GSC patients are needed to allow not only testing the repeatability of our findings, but also investigation of relationships of specific mitochondrial protein deficiencies or excesses and concurrent cytokine, genetic, or other factors to GDD's neurological and cognitive symptoms. Studies of neuronal mitochondrial proteins as diagnostic markers or indicators of treatment effectiveness are also warranted., Competing Interests: The 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 © 2022 Goetzl, Maecker, Rosenberg-Hasson and Koran.)

Background: COVID-19 caused by SARS-CoV-2 ranges from asymptomatic to severe disease and can cause fatal and devastating outcome in many cases. In this study, we have compared the clinical, biochemical and immunological parameters across the different disease spectrum of COVID-19 in Bangladeshi patients., Methodology/principal Findings: This longitudinal study was conducted in two COVID-19 hospitals and also around the community in Dhaka city in Bangladesh between November 2020 to March 2021. A total of 100 patients with COVID-19 infection were enrolled and classified into asymptomatic, mild, moderate and severe cases (n = 25/group). In addition, thirty age and sex matched healthy participants were enrolled and 21 were analyzed as controls based on exclusion criteria. After enrollment (study day1), follow-up visits were conducted on day 7, 14 and 28 for the cases. Older age, male gender and co-morbid conditions were the risk factors for severe COVID-19 disease. Those with moderate and severe cases of infection had low lymphocyte counts, high neutrophil counts along with a higher neutrophil-lymphocyte ratio (NLR) at enrollment; this decreased to normal range within 42 days after the onset of symptom. At enrollment, D-dimer, CRP and ferritin levels were elevated among moderate and severe cases. The mild, moderate, and severe cases were seropositive for IgG antibody by day 14 after enrollment. Moderate and severe cases showed significantly higher IgM and IgG levels of antibodies to SARS-CoV-2 compared to mild and asymptomatic cases., Conclusion/significance: We report on the clinical, biochemical, and hematological parameters associated with the different severity of COVID-19 infection. We also show different profile of antibody response against SARS-CoV-2 in relation to disease severity, especially in those with moderate and severe disease manifestations compared to the mild and asymptomatic infection., Competing Interests: The authors have declared that no competing interests exist.