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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.

Sepsis poses a significant global health challenge due to immune system dysregulation. This narrative review explores the complex relationship between antibiotics and the immune system, aiming to clarify the involved mechanisms and their clinical impacts. From pre-clinical studies, antibiotics exhibit various immunomodulatory effects, including the regulation of pro-inflammatory cytokine production, interaction with Toll-Like Receptors, modulation of the P38/Pmk-1 Pathway, inhibition of Matrix Metalloproteinases, blockade of nitric oxide synthase, and regulation of caspase-induced apoptosis. Additionally, antibiotic-induced alterations to the microbiome are associated with changes in systemic immunity, affecting cellular and humoral responses. The adjunctive use of antibiotics in sepsis patients, particularly macrolides, has attracted attention due to their immune-regulatory effects. However, there are limited data comparing different types of macrolides. More robust evidence comes from studies on community-acquired pneumonia, especially in severe cases with a hyper-inflammatory response. While studies on septic shock have shown mixed results regarding mortality rates and immune response modulation, conflicting findings are also observed with macrolides in acute respiratory distress syndrome. In conclusion, there is a pressing need to tailor antibiotic therapy based on the patient's immune profile to optimize outcomes in sepsis management.

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., (© 2024. The Author(s).)

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.

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., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: The aim of this randomized clinical trial (RCT) was to compare immunological changes in virally suppressed people living with HIV (PLWH) switching from a three-drug regimen (3DR) to a two-drug regimen (2DR)., Methods: An open-label, prospective RCT enrolling PLWH receiving a 3DR who switched to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) or dolutegravir/lamivudine (DTG/3TC) was performed. Blood was taken at baseline and months 6 and 12. The primary outcome was the change in CD4+ or CD8+ T-cell counts and CD4/CD8 ratio over time points. The secondary outcomes were the changes in immunological and inflammatory parameters. Parametric mixed-linear models with random intercepts and slopes were fitted separately for each marker after controlling for potential confounders., Results: Between the two arms (33 PLWH each), there was no difference in CD4+ or CD8+ T cells, CD4/CD8 ratio, and IL-6 trajectories. PLWH switching to DTG/3TC had increased levels of both transitional memory and terminally differentiated CD4+ T cells (arm-time interaction p-value = 0.02) and to a lesser extent for the corresponding CD8+ T-cell subsets (p = 0.09). Significantly lower levels of non-classical monocytes were detected in the B/F/TAF arm at T6 (diff = -6.7 cells/mm 3 ; 95% CI; -16, +2.6; p-value for interaction between arm and time = 0.03). All differences were attenuated at T12., Conclusion: No evidence for a difference in absolute CD4+ and CD8+ T-cell counts, CD4/CD8 ratio, and IL-6 trajectories by study arm over 12 months was found. PLWH on DTG/3TC showed higher levels of terminally differentiated and exhausted CD4+ and CD8+ T lymphocytes and non-classical monocytes at T6. Further studies are warranted to better understand the clinical impact of our results., Clinical Trial Registration: https://clinicaltrials.gov, identifier NCT04054089., Competing Interests: GG and CM received a research grant and a speaker honorarium from Gilead, ViiV, MERCK, and Jansen. GG and CM are on the advisory boards of Gilead, ViiV, and MERCK. JM received a speaker honorarium from Gilead and ViiV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cossarizza, Cozzi-Lepri, Mattioli, Paolini, Neroni, De Biasi, Tartaro, Borella, Fidanza, Gibellini, Beghetto, Roncaglia, Nardini, Milic, Menozzi, Cuomo, Digaetano, Orlando, Borghi, Guaraldi and Mussini.)

In preclinical studies rapamycin was found to target neuroinflammation, by expanding regulatory T cells, and affecting autophagy, two pillars of amyotrophic lateral sclerosis (ALS) pathogenesis. Herein we report a multicenter, randomized, double-blind trial, in 63 ALS patients who were randomly assigned in a 1:1:1 ratio to receive rapamycin 2 mg/m 2 /day,1 mg/m 2 /day or placebo (EUDRACT 2016-002399-28; NCT03359538). The primary outcome, the number of patients exhibiting an increase >30% in regulatory T cells from baseline to treatment end, was not attained. Secondary outcomes were changes from baseline of T, B, NK cell subpopulations, inflammasome mRNA expression and activation status, S6-ribosomal protein phosphorylation, neurofilaments; clinical outcome measures of disease progression; survival; safety and quality of life. Of the secondary outcomes, rapamycin decreased mRNA relative expression of the pro-inflammatory cytokine IL-18, reduced plasmatic IL-18 protein, and increased the percentage of classical monocytes and memory switched B cells, although no corrections were applied for multiple tests. In conclusion, we show that rapamycin treatment is well tolerated and provides reassuring safety findings in ALS patients, but further trials are necessary to understand the biological and clinical effects of this drug in ALS., (© 2023. Springer Nature Limited.)

Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

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., 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 © 2023 De Biasi, Mattioli, Meschiari, Lo Tartaro, Paolini, Borella, Neroni, Fidanza, Busani, Girardis, Coppi, Mattioli, Guaraldi, Mussini, Cossarizza and Gibellini.)

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., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: An unprecedented global monkeypox outbreak started in May, 2022. No data are yet available about the dynamics of the immune response against monkeypox virus. The aim of this study was to describe kinetics of T-cell response, inflammatory profile, and pox-specific T-cell induction in patients with laboratory-confirmed monkeypox., Methods: 17 patients with laboratory-confirmed monkeypox admitted at the Lazzaro Spallanzani National Institute for Infectious Diseases (Rome, Italy), from May 19, to July 7, 2022, were tested for differentiation and activation profile of CD4 and CD8 T (expression of CD38, PD-1, and CD57 assessed by flow cytometry), frequency of pox-specific T cells (by standard interferon-γ ELISpot), and release of interleukin (IL)-1β, IL-6, IL-8, and tumour necrosis factor (TNF) in plasma (by ELISA). All patients were tested 10-12 days after symptoms onset. In a subgroup of nine patients with a laboratory-confirmed monkeypox, the kinetics of the immune response were analysed longitudinally according to timing from symptoms onset and compared with ten healthy donors (ie, health-care workers recruited from the same institution)., Findings: Among the 17 patients, ten were HIV negative and seven HIV positive, all with good viro-immunological status. On days 0-3 from symptom onset, patients with laboratory-confirmed monkeypox were characterised by a statistically significant reduction in CD4+ T cells (p=0·0011) and a concurrent increase of CD8+ T cells (p=0·0057) compared with healthy donors. A lower proportion of naive (CD45RA+CD27+) CD4+ T cells was observed in six (67%) of nine patients and a concomitant higher proportion of effector memory (CD45RA-CD27-) CD4+ T cells in all patients; this skewed immune profile tended to normalise over time. A similar differentiated profile was also observed in CD8+ T cells with a consistent expansion of terminally differentiated CD8+ T cells. Patients with monkeypox had a higher proportion of CD4+CD38+ and CD38+CD8+ T-cells than healthy donors, which normalised after 12-20 days from symptom onset. The expression of PD-1 and CD57 on CD4+ and CD8+ T-cells showed kinetics similar to that observed for CD38. Furthermore, the inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF) were higher in patients with monkeypox than in healthy donors and, although they decreased over time, they remained elevated after recovery. Almost all patients (15 [94%] of 16) developed a pox-specific Th1 response. No differences in immune cells profile were observed between patients with and without HIV, whereas paucysimptomatic patients (without systemic symptoms, with less than five skin lesions, and no other mucosal localisation of monkeypox) showed a less perturbed immune profile early after symptom onset., Interpretation: Our data showed the immunological signature of monkeypox virus infection, characterised by an early expansion of activated effector CD4+ and CD8+ T cells that persisted over time. Almost all patients, even regardless of HIV infection, developed a poxvirus-specific Th1 cell response. These results might have implications on the expected immunogenicity of monkeypox vaccination, suggesting that it might not be necessary to vaccinate people who have already been infected., Funding: Italian Ministry of Health., Translation: For the Italian translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

BackgroundUnderstanding the epidemiology of reinfections is crucial for SARS-CoV-2 control over a long period.AimTo evaluate the risk of SARS-CoV-2 reinfection by vaccination status, predominant variant and time after first infection.MethodsWe conducted a cohort study including all residents in the Reggio Emilia province on 31 December 2019, followed up until 28 February 2022 for SARS-CoV-2 first infection and reinfection after 90 days. Cox models were used to compare risk of first infection vs reinfection, adjusting for age, sex, vaccine doses and comorbidities.ResultsThe cohort included 538,516 residents, 121,154 with first SARS-CoV-2 infections and 3,739 reinfections, most in the Omicron BA.1 period. In the pre-Omicron period, three doses of vaccine reduced risk of reinfection by 89% (95% CI: 87-90), prior infection reduced risk by 90% (95% CI: 88-91), while two doses and infection reduced risk by 98% (95% CI: 96-99). In the Omicron BA.1 period, protection estimates were 53% (95% CI: 52-55), 9% (95% CI: 4-14) and 76% (95% CI: 74-77). Before Omicron, protection from reinfection remained above 80% for up to 15 months; with Omicron BA.1, protection decreased from 71% (95% CI: 65-76) at 5 months to 21% (95% CI: 10-30) at 22 months from the first infection. Omicron BA.1 reinfections showed 48% (95% CI: 10-57) lower risk of severe disease than first infections.ConclusionsNatural immunity acquired with previous variants showed low protection against Omicron BA.1. Combined vaccination and natural immunity seems to be more protective against reinfection than either alone. Vaccination of people with prior infection reduced the risk of severe disease.

Background: Human dental pulp stem cells represent a mesenchymal stem cell niche localized in the perivascular area of dental pulp and are characterized by low immunogenicity and immunomodulatory/anti-inflammatory properties. Pericytes, mural cells surrounding the endothelium of small vessels, regulate numerous functions including vessel growth, stabilization and permeability. It is well established that pericytes have a tight cross talk with endothelial cells in neoangiogenesis and vessel stabilization, which are regulated by different factors, i.e., microenvironment and flow-dependent shear stress. The aim of this study was to evaluate the effects of a pulsatile unidirectional flow in the presence or not of an inflammatory microenvironment on the biological properties of pericyte-like cells isolated from human dental pulp (hDPSCs)., Methods: Human DPSCs were cultured under both static and dynamic conditions with or without pre-activated peripheral blood mononuclear cells (PBMCs). Pulsatile unidirectional flow shear stress was generated by using a specific peristaltic pump. The angiogenic potential and inflammatory properties of hDPSCs were evaluated through reverse phase protein microarrays (RPPA), confocal immunofluorescence and western blot analyses., Results: Our data showed that hDPSCs expressed the typical endothelial markers, which were up-regulated after endothelial induction, and were able to form tube-like structures. RPPA analyses revealed that these properties were modulated when a pulsatile unidirectional flow shear stress was applied to hDPSCs. Stem cells also revealed a downregulation of the immune-modulatory molecule PD-L1, in parallel with an up-regulation of the pro-inflammatory molecule NF-kB. Immune-modulatory properties of hDPSCs were also reduced after culture under flow-dependent shear stress and exposure to an inflammatory microenvironment. This evidence was strengthened by the detection of up-regulated levels of expression of pro-inflammatory cytokines in PBMCs., Conclusions: In conclusion, the application of a pulsatile unidirectional flow shear stress induced a modulation of immunomodulatory/inflammatory properties of dental pulp pericyte-like cells., (© 2023. The Author(s).)

The formation of a robust long-term antigen (Ag)-specific memory, both humoral and cell-mediated, is created following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. Here, by using polychromatic flow cytometry and complex data analyses, we deeply investigated the magnitude, phenotype, and functionality of SARS-CoV-2-specific immune memory in two groups of healthy subjects after heterologous vaccination compared to a group of subjects who recovered from SARS-CoV-2 infection. We find that coronavirus disease 2019 (COVID-19) recovered patients show different long-term immunological profiles compared to those of donors who had been vaccinated with three doses. Vaccinated individuals display a skewed T helper (Th)1 Ag-specific T cell polarization and a higher percentage of Ag-specific and activated memory B cells expressing immunoglobulin (Ig)G compared to those of patients who recovered from severe COVID-19. Different polyfunctional properties characterize the two groups: recovered individuals show higher percentages of CD4 + T cells producing one or two cytokines simultaneously, while the vaccinated are distinguished by highly polyfunctional populations able to release four molecules, namely, CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF), and interleukin (IL)-2. These data suggest that functional and phenotypic properties of SARS-CoV-2 adaptive immunity differ in recovered COVID-19 individuals and vaccinated ones., 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 reviewer AC declared a past co-authorship with some of the authors to the handling editor., (Copyright © 2023 Lo Tartaro, Paolini, Mattioli, Swatler, Neroni, Borella, Santacroce, Di Nella, Gozzi, Busani, Cuccorese, Trenti, Meschiari, Guaraldi, Girardis, Mussini, Piwocka, Gibellini, Cossarizza and De Biasi.)

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.

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., (© 2023 Wiley Periodicals LLC.)

Lung cancer is the leading cancer in the world, accounting for 1.2 million of new cases annually, being responsible for 17.8% of all cancer deaths. In particular, non-small cell lung cancer (NSCLC) is involved in approximately 85% of all lung cancers with a high lethality probably due to the asymptomatic evolution, leading patients to be diagnosed when the tumor has already spread to other organs. Despite the introduction of new therapies, which have improved the long-term survival of these patients, this disease is still not well cured and under controlled. Over the past two decades, single-cell technologies allowed to deeply profile both the phenotypic and metabolic aspects of the immune cells infiltrating the TME, thus fostering the identification of predictive biomarkers of prognosis and supporting the development of new therapeutic strategies. In this review, we discuss phenotypic and functional characteristics of the main subsets of tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating myeloid cells (TIMs) that contribute to promote or suppress NSCLC development and progression. We also address two emerging aspects of TIL and TIM biology, i.e., their metabolism, which affects their effector functions, proliferation, and differentiation, and their capacity to interact with cancer stem cells., 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 Aramini, Masciale, Samarelli, Dubini, Gaudio, Stella, Morandi, Dominici, De Biasi, Gibellini and Cossarizza.)

Several studies have identified main changes in T- and B-lymphocyte subsets during chronic HIV infection, but few data exist on how these subsets behave during the initial phase of HIV infection. We enrolled 22 HIV-infected patients during the acute stage of infection before the initiation of antiretroviral therapy (ART). Patients had blood samples drawn previous to ART initiation (T0), and at 2 (T1) and 12 (T2) months after ART initiation. We quantified cellular HIV-DNA content in sorted naïve and effector memory CD4 T cells and identified the main subsets of T- and B-lymphocytes using an 18-parameter flow cytometry panel. We identified correlations between the patients' clinical and immunological data using PCA. Effective HIV treatment reduces integrated HIV DNA in effector memory T cells after 12 months (T2) of ART. The main changes in CD4+ T cells occurred at T2, with a reduction of activated memory, cytolytic and activated/exhausted stem cell memory T (T SCM ) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted T SCM . At T2 a reduction of plasmablasts and exhausted B cells was also observed. A negative correlation was found between the total CD4+ T-cell count and IgM-negative plasmablasts. In patients initiating ART immediately following acute/early HIV infection, the fine analysis of T- and B-cell subsets has allowed us to identify and follow main modifications due to effective treatment, and to identify significant changes in CD4+ and CD8+ T memory stem cells.

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.

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., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress., (© 2022. The Author(s).)

Aging is a major risk factor for developing severe COVID-19, but few detailed data are available concerning immunological changes after infection in aged individuals. Here we describe main immune characteristics in 31 patients with severe SARS-CoV-2 infection who were >70 years old, compared to 33 subjects <60 years of age. Differences in plasma levels of 62 cytokines, landscape of peripheral blood mononuclear cells, T cell repertoire, transcriptome of central memory CD4 + T cells, specific antibodies are reported along with features of lung macrophages. Elderly subjects have higher levels of pro-inflammatory cytokines, more circulating plasmablasts, reduced plasmatic level of anti-S and anti-RBD IgG3 antibodies, lower proportions of central memory CD4 + T cells, more immature monocytes and CD56 + pro-inflammatory monocytes, lower percentages of circulating follicular helper T cells (cTfh), antigen-specific cTfh cells with a less activated transcriptomic profile, lung resident activated macrophages that promote collagen deposition and fibrosis. Our study underlines the importance of inflammation in the response to SARS-CoV-2 and suggests that inflammaging, coupled with the inability to mount a proper anti-viral response, could exacerbate disease severity and the worst clinical outcome in old patients., (© 2022. The Author(s).)

Several COVID-19 convalescents suffer from the post-acute COVID-syndrome (PACS)/long COVID, with symptoms that include fatigue, dyspnea, pulmonary fibrosis, cognitive dysfunctions or even stroke. Given the scale of the worldwide infections, the long-term recovery and the integrative health-care in the nearest future, it is critical to understand the cellular and molecular mechanisms as well as possible predictors of the longitudinal post-COVID-19 responses in convalescent individuals. The immune system and T cell alterations are proposed as drivers of post-acute COVID syndrome. However, despite the number of studies on COVID-19, many of them addressed only the severe convalescents or the short-term responses. Here, we performed longitudinal studies of mild, moderate and severe COVID-19-convalescent patients, at two time points (3 and 6 months from the infection), to assess the dynamics of T cells immune landscape, integrated with patients-reported symptoms. We show that alterations among T cell subsets exhibit different, severity- and time-dependent dynamics, that in severe convalescents result in a polarization towards an exhausted/senescent state of CD4+ and CD8+ T cells and perturbances in CD4+ Tregs. In particular, CD8+ T cells exhibit a high proportion of CD57+ terminal effector cells, together with significant decrease of naïve cell population, augmented granzyme B and IFN-γ production and unresolved inflammation 6 months after infection. Mild convalescents showed increased naïve, and decreased central memory and effector memory CD4+ Treg subsets. Patients from all severity groups can be predisposed to the long COVID symptoms, and fatigue and cognitive dysfunctions are not necessarily related to exhausted/senescent state and T cell dysfunctions, as well as unresolved inflammation that was found only in severe convalescents. In conclusion, the post-COVID-19 functional remodeling of T cells could be seen as a two-step process, leading to distinct convalescent immune states at 6 months after infection. Our data imply that attenuation of the functional polarization together with blocking granzyme B and IFN-γ in CD8+ cells might influence post-COVID alterations in severe convalescents. However, either the search for long COVID predictors or any treatment to prevent PACS and further complications is mandatory in all patients with SARS-CoV-2 infection, and not only in those suffering from severe COVID-19., 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 Wiech, Chroscicki, Swatler, Stepnik, De Biasi, Hampel, Brewinska-Olchowik, Maliszewska, Sklinda, Durlik, Wierzba, Cossarizza and Piwocka.)

Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Although it is now widely accepted that host inflammatory response contributes to COVID-19 immunopathogenesis, the pathways and mechanisms driving disease severity and clinical outcome remain poorly understood. In the effort to identify key soluble mediators that characterize life-threatening COVID-19, we quantified 62 cytokines, chemokines and other factors involved in inflammation and immunity in plasma samples, collected at hospital admission, from 80 hospitalized patients with severe COVID-19 disease who were stratified on the basis of clinical outcome (mechanical ventilation or death by day 28). Our data confirm that age, as well as neutrophilia, lymphocytopenia, procalcitonin, D-dimer and lactate dehydrogenase are strongly associated with the risk of fatal COVID-19. In addition, we found that cytokines related to TH2 regulations (IL-4, IL-13, IL-33), cell metabolism (lep, lep-R) and interferons (IFNα, IFNβ, IFNγ) were also predictive of life-threatening COVID-19., 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 Gibellini, De Biasi, Meschiari, Gozzi, Paolini, Borella, Mattioli, Lo Tartaro, Fidanza, Neroni, Busani, Girardis, Guaraldi, Mussini, Cozzi-Lepri and Cossarizza.)

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., (© 2021 Wiley-VCH GmbH.)

Latent Mycobacterium tuberculosis infection (LTBI) and active tuberculosis in prisoners are higher than the general population and are two public health concerns, especially in low- and middle-income countries. We conducted a cross-sectional study to determine the prevalence and the factors associated with LTBI among the inmate population detained in three Southern Italian penitentiaries. Tuberculin intradermal reaction skin test was performed on the inmates who agreed to participate in the study. In case of positivity, the QuantiFERON-TB test was performed. In those positive to QuantiFERON, chest X-ray films were performed, and treatment initiated. A total of 381 inmates accepted to participate. The prevalence of LTBI was 4.2%. In the analysis, LTBI was associated with no self-reported contact with active tuberculosis patients within the prisons, and 10% of subjects admitted the use of inhaled drugs. No HIV coinfections were found. No cases of active symptomatic tuberculosis were identified during the study period. Our results confirm that incarceration increases the risk of tuberculous infection. Non-EU nationality and a history of drug addiction appear to be major risk factors for tuberculosis infection in the penitentiary setting. Reinforcing tuberculosis control is essential to prevent its transmission in prisons.

Background: Extracellular vesicles (EVs) are a heterogeneous family of small vesicles released by donor cells and absorbed by recipient cells, which represent important mediators with fundamental roles in both physiological and pathological conditions. EVs are present in a large variety of biological fluids and have a great diagnostic and prognostic value. They have gained the interest of the scientific community due to their extreme versatility. In fact, they allow us to hypothesize new therapeutic strategies since, in addition to being cell signal mediators, they play an important role as biomarkers, drug vehicles, and potential new therapeutic agents. They are also involved in immunoregulation, have the ability to transmit resistance to a drug from one cell to a more sensitive one, and can act as drug delivery systems., Objective: The main reciprocal interactions between EVs and immunosuppressive drugs will be presented., Results: The known interactions between EVs and immunosuppressive drugs, in particular cyclosporin, glucocorticoids, rapamycin, methotrexate, cyclophosphamide, eculizumab, infliximab, certolizumab, etanercept, glatiramer acetate, and fingolimod are presented., Conclusion: This review provides relevant information on the links between EVs and immunosuppressive drugs with a focus on EVs' role as tools to assess the effects of immunosuppressants, suggesting innovative properties and new possible therapeutic uses., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

The altered numbers and functions of cells belonging to immunoregulatory cell networks such as T regulatory (Tregs) and invariant Natural Killer T (iNKT) cells have been reported in Multiple Sclerosis (MS), an immune-mediated disease. We aimed to assess the frequencies of Tregs and iNKT cells in MS patients throughout a one-year treatment with fingolimod (FTY) and to correlate immunological data with efficacy and safety data. The percentage of Tregs (defined as Live Dead-CD3 + CD4 + FoxP3 + CD25++/CD127- cells) increased steadily throughout the year, while there was no significant difference in the absolute number or percentage of iNKT cells (defined as CD3 + CD14-CD19- Vα24-Jα18 TCR+ cells). However, out of all the iNKT cells, the CD8+ iNKT and CD4-CD8- double-negative (DN) cell percentages steadily increased, while the CD4+ iNKT cell percentages decreased significantly. The mean percentage of CD8+ T cells at all time-points was lower in patients with infections throughout the study. The numbers and percentages of DN iNKT cells were more elevated, considering all time-points, in patients who presented a clinical relapse. FTY may, therefore, exert its beneficial effect in MS patients through various mechanisms, including the increase in Tregs and in iNKT subsets with immunomodulatory potential such as CD8+ iNKT cells. The occurrence of infections was associated with lower mean CD8+ cell counts during treatment with FTY.

SARS-CoV-2 infection can affect all human beings, including pregnant women. Thus, understanding the immunological changes induced by the virus during pregnancy is nowadays of pivotal importance. Here, using peripheral blood from 14 pregnant women with asymptomatic or mild SARS-CoV-2 infection, we investigate cell proliferation and cytokine production, measure plasma levels of 62 cytokines, and perform a 38-parameter mass cytometry analysis. Our results show an increase in low density neutrophils but no lymphopenia or gross alterations of white blood cells, which display normal levels of differentiation, activation or exhaustion markers and show well preserved functionality. Meanwhile, the plasma levels of anti-inflammatory cytokines such as interleukin (IL)-1RA, IL-10 and IL-19 are increased, those of IL-17, PD-L1 and D-dimer are decreased, but IL-6 and other inflammatory molecules remain unchanged. Our profiling of antiviral immune responses may thus help develop therapeutic strategies to avoid virus-induced damages during pregnancy., (© 2021. The Author(s).)

Cell death mechanisms are crucial to maintain an appropriate environment for the functionality of healthy cells. However, during viral infections, dysregulation of these processes can be present and can participate in the pathogenetic mechanisms of the disease. In this review, we describe some features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some immunopathogenic mechanisms characterizing the present coronavirus disease (COVID-19). Lymphopenia and monocytopenia are important contributors to COVID-19 immunopathogenesis. The fine mechanisms underlying these phenomena are still unknown, and several hypotheses have been raised, some of which assign a role to cell death as far as the reduction of specific types of immune cells is concerned. Thus, we discuss three major pathways such as apoptosis, necroptosis, and pyroptosis, and suggest that all of them likely occur simultaneously in COVID-19 patients. We describe that SARS-CoV-2 can have both a direct and an indirect role in inducing cell death. Indeed, on the one hand, cell death can be caused by the virus entry into cells, on the other, the excessive concentration of cytokines and chemokines, a process that is known as a COVID-19-related cytokine storm, exerts deleterious effects on circulating immune cells. However, the overall knowledge of these mechanisms is still scarce and further studies are needed to delineate new therapeutic strategies.

Monocyte Distribution Width (MDW), a new cytometric parameter correlating with cytomorphologic changes occurring upon massive monocyte activation, has recently emerged as promising early biomarker of sepsis. Similar to sepsis, monocyte/macrophage subsets are considered key mediators of the life-threatening hyper-inflammatory disorder characterizing severe COVID-19. In this study, we longitudinally analyzed MDW values in a cohort of 87 COVID-19 patients consecutively admitted to our hospital, showing significant correlations between MDW and common inflammatory markers, namely CRP (p < 0.001), fibrinogen (p < 0.001) and ferritin (p < 0.01). Moreover, high MDW values resulted to be prognostically associated with fatal outcome in COVID-19 patients (AUC = 0.76, 95% CI: 0.66-0.87, sensitivity 0.75, specificity 0.70, MDW threshold 26.4; RR = 4.91, 95% CI: 1.73-13.96; OR = 7.14, 95% CI: 2.06-24.71). This pilot study shows that MDW can be useful in the monitoring of COVID-19 patients, as this innovative hematologic biomarker is: (1) easy to obtain, (2) directly related to the activation state of a fundamental inflammatory cell subset (i.e. monocytes, pivotal in both cytokine storm and sepsis immunopathogenesis), (3) well correlated with clinical severity of COVID-19-associated inflammatory disorder, and, in turn, (4) endowed with relevant prognostic significance. Additional studies are needed to define further the clinical impact of MDW testing in the management of COVID-19 patients.

The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8 + T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8 + T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8 + T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8 + T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8 + T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8 + T cells during convalescence.

Background: Systemic immune activation and inflammation in chronic HIV infection are driving factors of non-AIDS-related events, including neurocognitive impairment. The role of inflammasome in monocytes from patients with HIV infection has been extensively studied, but its association with the extent of neurocognitive dysfunction has been poorly investigated., Methods: We enrolled 79 HIV-positive patients; 44 with varying levels of HIV-associated neurocognitive disorder (HAND) and 35 without and 8 healthy donors. HAND subtypes included asymptomatic neurocognitive impairment (asymptomatic neurocognitive impairment; n = 19), mild neurocognitive disorder (MND; n = 17), and HIV-associated dementia (n = 8). We quantified plasmatic concentrations of proinflammatory cytokines (TNF-α, IL-6, IL-17A, IL-1β, and IFN-γ) for all HIV patients, and the mRNA expression of genes involved in the inflammasome activity (NLRP3, PYCARD, NAIP, AIM2, IL-1β, and IL-18) in monocytes of a subgroup of 28 HIV patients and 8 healthy donors., Results: HIV patients' plasma concentrations of IFN-γ, IL-1β, and IL-17A were undetectable. Levels of TNF-α and IL-6 were similar among the HIV patient groups. A trend toward an increased expression of inflammasome genes according to neurocognitive disorder severity was observed. Of note, the NLRP3 mRNA relative expression was higher in MND compared with other groups, and IL-1β was lower in MND than HIV-associated dementia patients., Conclusions: Changes in inflammasome components in circulating monocytes according to different HAND severity suggest that NLRP3 may be a possible biomarker or target to better understand and treat the link between systemic inflammation and neurocognitive impairment in HIV infection., Competing Interests: The Iranian Research Center for HIV/AIDS (Tehran University of Medical Sciences) provided a fellowship to E.M.-T. and the International Society for Advancement of Cytometry (ISAC) the Marylou Ingram Scholarship to S.D.-B. The remaining authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8 + T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8 + T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8 + population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

A major breakthrough in cancer immunotherapy was the development of monoclonal antibodies targeting inhibitory immune checkpoint proteins. This approach demonstrated significant antitumor activity and efficacy in different cancer types, including metastatic renal cell carcinoma (mRCC). In the majority of patients, this drug is able to restore the patient's tumour-specific T-cell-mediated response thus improving both overall survival and objective response rate. However, a lack of clinical response occurs in a number of patients, raising questions about how to predict and increase the number of patients who receive long-term clinical benefit from immune checkpoint therapy or not. The aim of this review is to summarize available data about immune biomarkers in patients with mRCC treated with immunotherapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Septic shock still has a high mortality rate which has not hinted at decreasing in recent years. Unfortunately, randomized trials failed mainly because the septic patient was considered as a homogeneous entity. All this creates a sort of therapeutic impotence in everyday clinical practice in treating patients with septic shock. The need to customize therapy on each patient with sepsis has now become an established necessity. In this scenario, adjuvant therapies can help if interpreted as modulators of the immune system. Indeed, the host's immune response differs from patient to patient based on the virulence of the pathogen, comorbidity, infection site, and prolonged hospitalization. In this review, we summarize the rationale for using immunoglobulins as an adjunctive treatment. Furthermore, we would like to suggest a possible protocol to personalize treatment in the different clinical scenarios of the host's response to serious infectious events., Competing Interests: MG has consulted for Biotest-Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Busani, Roat, Tosi, Biagioni, Coloretti, Meschiari, Gelmini, Brugioni, De Biasi and Girardis.)

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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

During the last decade, the rapid progress in the development of next-generation sequencing (NGS) technologies has provided relevant insights into complex biological systems, ranging from cancer genomics to microbiology. Among NGS technologies, single-cell RNA sequencing is currently used to decipher the complex heterogeneity of several biological samples, including T cells. Even if this technique requires specialized equipment and expertise, nowadays it is broadly applied in research. In this chapter, we will provide an optimized protocol for the isolation of T cells and the preparation of RNA sequencing libraries by using droplet digital technology (ddSEQ, Bio-Rad Laboratories). We will also illustrate a guide to the main steps of data processing and options for data interpretation. This protocol will support users in building a single-cell experimental framework, from sample preparation to data interpretation.

: In the last years, novel, exciting immunological findings of interest for HIV research and treatment were identified thanks to different cytometric approaches. The analysis of the phenotypes and functionality of cells belonging to the immune system could clarify their role in the immunopathogenesis of HIV infection, and to elaborate key concepts, relevant in the treatment of this disease. Important discoveries have been made concerning cells that are important for protective immunity like lymphocytes that display polyfunctionality, resident memory T cells, innate lymphoid cells, to mention a few. The complex phenotype of myeloid-derived suppressor cells has been investigated, and relevant changes have been reported during chronic and primary HIV infection, in correlation with changes in CD4 T-cell number, T-cell activation, and with advanced disease stage. The search for markers of HIV persistence present in latently infected cells, namely those molecules that are important for a functional or sterilizing cure, evidenced the role of follicular helper T cells, and opened a discussion on the meaning and use of different surface molecules not only in identifying such cells, but also in designing new strategies. Finally, advanced technologies based upon the simultaneous detection of HIV-RNA and proteins at the single cell level, as well as those based upon spectral cytometry or mass cytometry are now finding new actors and depicting a new scenario in the immunopathogenesis of the infection, that will allow to better design innovative therapies based upon novel drugs and vaccines.

The study aimed to identify the effects of whole-body cryotherapy (WBC) on immunological, hormonal, and metabolic responses of non-professional male athletes. Ten cyclists and ten middle-distance runners received 3 once-a-day sessions of WBC. Before initiating and after the final WBC session, a full set of hematologic parameters, serum chemistry profile, hormones, circulating mitochondrial (mt) DNA levels, cytokines, and chemokines concentration were evaluated. The phenotype of monocyte, T cells, and B cells was analyzed. mRNA expression of 6 genes involved in inflammasome activation (NAIP, AIM2, NLRP3, PYCARD, IL-1β, and IL-18) was quantified. WBC reduced glucose and C and S protein and increased HDL, urea, insulin-like growth factor (IGF)-1, follicle-stimulating hormone, IL-18, IL-1RA, CCL2, and CXCL8. Intermediate and non-classical monocyte percentages decreased, and the CD14, CCR5, CCR2, and CXCR4 expressions changed in different subsets. Only IL-1β mRNA increased in monocytes. Finally, a redistribution of B and T cell subsets was observed, suggesting the migration of mature cells to tissue. WBC seems to induce changes in both innate and adaptive branches of the immune system, hormones, and metabolic status in non-professional male athletes, suggesting a beneficial involvement of WBC in tissue repair.

Microbial translocation has been suggested as a major driver of chronic immune activation HIV infection. Thus, we compared the extent of microbial translocation in patients with acute HIV infection and patients followed after CD4-guided structured treatment interruption (STI) by measuring different circulating markers: (1) lipopolysaccharide (LPS)-binding protein (LBP), (2) bacterial DNA, (3) soluble CD14 (sCD14), and (4) mitochondrial DNA (mtDNA). Bacterial DNA and sCD14 levels were similar in all groups. Patients in acute phase showed higher levels of LBP and mtDNA. In STI, we found a positive correlation between the percentage of CD8+ T cells and bacterial DNA levels. Considering all patients, LBP was positively correlated with the percentage and the absolute count of CD8+ T cells, and with mtDNA stressing the importance of mitochondrial products in sustaining chronic immune activation.

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 O 2 - , 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., Competing Interests: Declaration of competing interest The authors have no competing interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© 2020 Wiley‐VCH GmbH.)

The immune system of patients infected by SARS-CoV-2 is severely impaired. Detailed investigation of T cells and cytokine production in patients affected by COVID-19 pneumonia are urgently required. Here we show that, compared with healthy controls, COVID-19 patients' T cell compartment displays several alterations involving naïve, central memory, effector memory and terminally differentiated cells, as well as regulatory T cells and PD1 + CD57 + exhausted T cells. Significant alterations exist also in several lineage-specifying transcription factors and chemokine receptors. Terminally differentiated T cells from patients proliferate less than those from healthy controls, whereas their mitochondria functionality is similar in CD4 + T cells from both groups. Patients display significant increases of proinflammatory or anti-inflammatory cytokines, including T helper type-1 and type-2 cytokines, chemokines and galectins; their lymphocytes produce more tumor necrosis factor (TNF), interferon-γ, interleukin (IL)-2 and IL-17, with the last observation implying that blocking IL-17 could provide a novel therapeutic strategy for COVID-19.

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., (© 2020 International Society for Advancement of Cytometry.)

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations of LONP1 have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in which Lonp1 was ablated. The homozygous Lonp -/- mouse was not vital, while the heterozygous Lonp1 wt/- showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) from Lonp1 wt/- mice showed a reduced expression of Lonp1 and Tfam , whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of the Lonp1 gene leads to impairment of mitochondrial ultrastructure and functions in vivo.