Healthy preterm newborns: Altered innate immunity and impaired monocyte function.

Birth prior to 37 completed weeks of gestation is referred to as preterm (PT). Premature newborns are at increased risk of developing infections as neonatal immunity is a developing structure. Monocytes, which are key players after birth, activate inflammasomes. Investigations into the identification of innate immune profiles in premature compared to full-term infants are limited. Our research includes the investigation of monocytes and NK cells, gene expression, and plasma cytokine levels to investigate any potential differences among a cohort of 68 healthy PT and full-term infants. According to high-dimensional flow cytometry, PT infants have higher proportions of CD56+/- CD16+ NK cells and immature monocytes, and lower proportions of classical monocytes. Gene expression revealed lower proportions of inflammasome activation after in vitro monocyte stimulation and the quantification of plasma cytokine levels expressed higher concentrations of alarmin S100A8. Our findings suggest that PT newborns have altered innate immunity and monocyte functional impairment, and pro-inflammatory plasmatic profile. This may explain PT infants' increased susceptibility to infectious disease and should pave the way for novel therapeutic strategies and clinical interventions.

Metabolic reprograming shapes neutrophil functions in severe COVID-19.

To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2, we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase, and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus, suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies.

A Comprehensive Analysis of Cytokine Network in Centenarians.

Cytokines have been investigated extensively in elderly people, with conflicting results. We performed a comprehensive analysis of the plasma levels of 62 cytokines and growth factors involved in the regulation of the immune system, in healthy centenarians, and middle-aged controls. We confirmed the previously observed increase in the levels of several pro-inflammatory cytokines, such as TNF-α and IL-6, and found that several other cytokines, directly or indirectly involved in inflammation (such as IFN-α, IL-23, CCL-5), were present at higher levels in centenarians. We did not observe any increase in the levels of anti-inflammatory cytokines, with the notable exception of the Th2-shifting cytokine IL-19. No relevant difference was observed in cytokines regulating T cell immunity. Several growth factors having a role in regulating immunity, such as G-CSF, GM-CSF, EGF, and VEGF, were upregulated in centenarians, too. Principal component analysis of the cytokine dataset showed that pro and anti-inflammatory cytokines were the variables that contributed the most to the variability of the data we observed.

Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy.

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment.

Patients Recovering from Severe COVID-19 Develop a Polyfunctional Antigen-Specific CD4+ T Cell Response.

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.

Efficient T-Cell Compartment in HIV-Positive Patients Receiving Orthotopic Liver Transplant and Immunosuppressive Therapy.

BACKGROUND: In patients undergoing orthotopic liver transplant (OLT), immunosuppressive treatment is mandatory and infections are leading causes of morbidity/mortality. Thus, it is essential to understand the functionality of cell-mediated immunity after OLT. The aim of the study was to identify changes in T-cell phenotype and polyfunctionality in human immunodeficiency virus-positive (HIV+) and -negative (HIV-) patients undergoing immunosuppressive treatment after OLT. METHODS: We studied peripheral blood mononuclear cells from 108 subjects divided into 4 groups of 27: HIV+ transplanted patients, HIV- transplanted patients, HIV+ nontransplanted patients, and healthy subjects. T-cell activation, differentiation, and cytokine production were analyzed by flow cytometry. RESULTS: Median age was 55 years (interquartile range, 52-59 years); the median CD4 count in HIV+ patients was 567 cells/mL, and all had undetectable viral load. CD4+ and CD8+ T-cell subpopulations showed different distributions between HIV+ and HIV- OLT patients. A cluster representing effector cells expressing PD1 was abundant in HIV- transplanted patients and they were characterized by higher levels of CD4+ T cells able to produce interferon-γ and tumor necrosis factor-α. CONCLUSIONS: HIV- transplanted patients have more exhausted or inflammatory T cells compared to HIV+ transplanted patients, suggesting that patients who have already experienced a form of immunosuppression due to HIV infection respond differently to anti-rejection therapy.

Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia.

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.

Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia.

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defense and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2-, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

Mitochondrial functionality and metabolism in T cells from progressive multiple sclerosis patients.

Patients with primary progressive (PP) and secondary progressive (SP) forms of multiple sclerosis (MS) exhibit a sustained increase in the number of Th1, T cytotoxic type-1 and Th17 cells in peripheral blood, suggesting that the progressive phase is characterized by a permanent peripheral immune activation. As T cell functionality and activation are strictly connected to their metabolic profile, we investigated the mitochondrial functionality and metabolic changes of T cell subpopulations in a cohort of progressive MS patients. T cells from progressive patients were characterized by low proliferation and increase of terminally differentiated/exhausted cells. T cells from PP patients showed lower Oxygen Consumption Rate and Extracellular Acidification Rate, lower mitochondrial mass, membrane potential and respiration than those of SP patients, a downregulation of transcription factors supporting respiration and higher tendency to shift towards glycolysis upon stimulation. Furthermore, PP effector memory T cells were characterized by higher Glucose transporter -1 levels and a higher expression of glycolytic-supporting genes if compared to SP patients. Overall, our data suggest that profound differences exist in the phenotypic and metabolic features of T cells from PP and SP patients, even though the two clinical phenotypes are considered part of the same disease spectrum.

Handling and Processing of Blood Specimens from Patients with COVID-19 for Safe Studies on Cell Phenotype and Cytokine Storm.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 heavily involves all those working in a laboratory. Samples from known infected patients or donors who are considered healthy can arrive, and a colleague might be asymptomatic but able to transmit the virus. Working in a clinical laboratory is posing several safety challenges. Few years ago, International Society for Advancement of Cytometry published guidelines to safely analyze and sort human samples that were revised in these days. We describe the procedures that we have been following since the first patient appeared in Italy, which have only slightly modified our standard one, being all human samples associated with risks. © 2020 International Society for Advancement of Cytometry.

Altered Expression of PYCARD, Interleukin 1ß, Interleukin 18, and NAIP in Successfully Treated HIV-Positive Patients With a Low Ratio of CD4+ to CD8+ T Cells.

The expression and activity of main inflammasome components in monocytes from successfully treated human immunodeficiency virus (HIV)-positive patients are poorly studied. Thus, we enrolled 18 patients with a low and 17 with a normal ratio of CD4+ T cells to CD8+ T cells and 11 healthy donors. We found that patients with a low ratio had decreased CCR2 expression among classical and intermediate monocytes and increased CCR5 expression among classical monocytes, compared with patients with a normal ratio. Patients with a low ratio also had higher NAIP and PYCARD messenger RNA levels after lipopolysaccharide stimulation, suggesting an altered ability to control immune activation that could affect their immune reconstitution.

Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR.

Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients.

Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.

Advances and Challenges in Sepsis Management: Modern Tools and Future Directions.

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population.

Cell metabolism: Functional and phenotypic single cell approaches.

Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.

SARS-CoV-2 Vaccination Responses in Anti-CD20-Treated Progressive Multiple Sclerosis Patients Show Immunosenescence in Antigen-Specific B and T Cells.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) shows that inflammation starts early and progresses with age. B cells play a central role in this process, contributing to cytokine production, defective regulatory functions, and abnormal immunoglobulin production, even in the central nervous system. Anti-CD20 (aCD20) therapies, which deplete CD20+ B cells, are largely used in the treatment of both relapsing remitting (RR) and progressive (PR) forms of MS. Although effective against MS symptoms and lesions detectable by magnetic resonance imaging, aCD20 therapies can reduce the immune response to COVID-19 vaccination. By using high-parameter flow cytometry, we examined the antigen-specific (Ag+) immune response six months post-third COVID-19 mRNA vaccination in MS patients with RR and PR forms on aCD20 therapy. Despite lower Ag+ B cell responses and lower levels of anti-SARS-CoV2, both total and neutralizing antibodies, RR and PR patients developed strong Ag+ T cell responses. We observed similar percentages and numbers of Ag+ CD4+ T cells and a high proportion of Ag+ CD8+ T cells, with slight differences in T cell phenotype and functionality; this, however, suggested the presence of differences in immune responses driven by age and disease severity.

Immunosenescence and vaccine efficacy revealed by immunometabolic analysis of SARS-CoV-2-specific cells in multiple sclerosis patients.

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments.

Analysis of Antigen-Specific T and B Cells for Monitoring Immune Protection Against SARS-CoV-2.

Immunological memory is the basis of protection against most pathogens. Long-living memory T and B cells able to respond to specific stimuli, as well as persistent antibodies in plasma and in other body fluids, are crucial for determining the efficacy of vaccination and for protecting from a second infection by a previously encountered pathogen. Antigen-specific cells are represented at a very low frequency in the blood, and indeed, they can be considered "rare events" present in the memory T-cell pool. Therefore, such events should be analyzed with careful attention. In the last 20 years, different methods, mostly based upon flow cytometry, have been developed to identify such rare antigen-specific cells, and the COVID-19 pandemic has given a dramatic impetus to characterize the immune response against the virus. In this regard, we know that the identification, enumeration, and characterization of SARS-CoV-2-specific T and B cells following infection and/or vaccination require i) the use of specific peptides and adequate co-stimuli, ii) the use of appropriate inhibitors to avoid nonspecific activation, iii) the setting of appropriate timing for stimulation, and iv) the choice of adequate markers and reagents to identify antigen-specific cells. Optimization of these procedures allows not only determination of the magnitude of SARS-CoV-2-specific responses but also a comparison of the effects of different combinations of vaccines or determination of the response provided by so-called "hybrid immunity," resulting from a combination of natural immunity and vaccine-generated immunity. Here, we present two methods that are largely used to monitor the response magnitude and phenotype of SARS-CoV-2-specific T and B cells by polychromatic flow cytometry, along with some tips that can be useful for the quantification of these rare events. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of antigen-specific T cells Basic Protocol 2: Identification of antigen-specific B cells.

Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab.

Introduction: A growing number of evidences suggest that the combination of hyperinflammation, dysregulated T and B cell response and cytokine storm play a major role in the immunopathogenesis of severe COVID-19. IL-6 is one of the main pro-inflammatory cytokines and its levels are increased during SARS-CoV-2 infection. Several observational and randomized studies demonstrated that tocilizumab, an IL-6R blocker, improves survival in critically ill patients both in infectious disease and intensive care units. However, despite transforming the treatment options for COVID-19, IL-6R inhibition is still ineffective in a fraction of patients. Methods: In the present study, we investigated the impact of two doses of tocilizumab in patients with severe COVID-19 who responded or not to the treatment by analyzing a panel of cytokines, chemokines and other soluble factors, along with the composition of peripheral immune cells, paying a particular attention to T and B lymphocytes. Results: We observed that, in comparison with non-responders, those who responded to tocilizumab had different levels of several cytokines and different T and B cells proportions before starting therapy. Moreover, in these patients, tocilizumab was further able to modify the landscape of the aforementioned soluble molecules and cellular markers. Conclusions: We found that tocilizumab has pleiotropic effects and that clinical response to this drug remain heterogenous. Our data suggest that it is possible to identify patients who will respond to treatment and that the administration of tocilizumab is able to restore the immune balance through the re-establishment of different cell populations affected by SARS-COV-2 infection, highlighting the importance of temporal examination of the pathological features from the diagnosis.