Evidence for mitochondrial Lonp1 expression in the nucleus

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

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

Innate immunity changes in soccer players after whole-body cryotherapy

Whole-body cryotherapy (WBC) consists of short exposure (up to 2-3 min) to dry air at cryogenic temperatures (up to -190 degrees C) and has recently been applied for muscle recovery after injury to reduce the inflammation process. We aimed to determine the impact of cryotherapy on immunological, hormonal, and metabolic responses in non-professional soccer players (NPSPs). Nine male NPSPs (age: 20 +/- 2 years) who trained regularly over 5 consecutive days, immediately before and after each training session, were subjected to WBC treatment (WBC-t). Blood samples were collected for the evaluation of fifty analytes including hematologic parameters, serum chemistry, and hormone profiles. Monocytes phenotyping (Mo) was performed and plasmatic markers, usually increased during inflammation [CCL2, IL-18, free mitochondrial (mt)DNA] or with anti-inflammatory effects (IL2RA, IL1RN), were quantified. After WBC-t, we observed reduced levels of ferritin, mean corpuscular hemoglobin, mean platelet volume, testosterone, and estradiol, which however remain within the normal ranges. The percentage of the total, intermediates and non-classical Mo increased, while classical Mo decreased. CXCR4 expression decreased in each Mo subset. Plasma IL18 and IL2RA levels decreased, while IL1RN only exhibited a tendency to decrease and CCL2 showed a tendency to increase. Circulating mtDNA levels were not altered following WBC-t. The differences observed in monocyte subsets after WBC-t may be attributable to their redistribution into the surrounding tissue. Moreover, the decrease of CXCR4 in Mo subpopulations could be coherent with their differentiation process. Thus, WBC through yet unknown mechanisms could promote their differentiation having a role in tissue repair

Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination

: 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

Melanoma metastatico e carcinoma renale: focus sul ruolo delle cellule T CD8+ nelle risposte agli inibitori dei checkpoint immunitari

I linfociti T CD8 giocano un ruolo centrale nell’immunità al cancro mediante la loro capacità di uccidere le cellule maligne. Tuttavia, la prolungata esposizione agli antigeni nel microambiente tumorale contribuisce a indurre un grave esaurimento delle cellule T CD8 (Tex). Durante gli ultimi due decenni il trattamento del cancro è stato rivoluzionato dagli inibitori dei checkpoint immunitari (ICIs), che bloccano l’attività dei recettori inibitori, come il PD-1, presenti sulla superfice delle cellule Tex, rinvigorendole. Nonostante l’osservazione di risposte durature alla terapia ICI, non tutti i pazienti rispondono al trattamento. Abbiamo focalizzato la nostra attenzione sull'identificazione delle alterazioni che si verificano nei linfociti T CD8 circolanti di pazienti con melanoma metastatico (mM) e carcinoma renale metastatico (mRCC) durante il trattamento con anti-PD1, al fine di capire come e perché alcuni pazienti rispondono o no alla terapia. La prima coorte comprendeva 28 pazienti mM, 17 dei quali definiti responsivi (R) mentre 11 non responsivi (NR) alla terapia. La seconda coorte comprendeva invece 19 pazienti mRCC, 5 dei quali definiti responsivi (R) mentre 14 non responsivi (NR). I PBMC crioconservati, ottenuti prima dell'inizio della terapia (T0) dopo il primo (T1), secondo (T2) e terzo ciclo di terapia (T3), sono stati studiati mediante citometria a flusso a 30 parametri in combinazione con il sequenziamento dell'RNA a singola cellula (scRNA-seq) e analizzate utilizzando FAUST, un nuovo metodo non parametrico per la scoperta non supervisionata di popolazioni cellulari. Nei pazienti R con mM abbiamo riscontrato un aumento delle cellule effettrici di memoria (EM) proliferanti esprimenti alti livelli di Ki67, ICOS, CD95, HLA-DR, CD71, CD98, CXCR6, granulisina e CD38, sia prima che dopo il primo e il secondo ciclo di terapia. L’scRNA-seq ha rivelato la presenza delle cellule T invarianti associate alla mucosa (MAIT) attivate esprimenti il CD69 e CXCR4 e il loro aumento nei pazienti R rispetto ai NR, sia prima e dopo la terapia con ICI. Insieme all'aumento in percentuale abbiamo anche osservato una maggiore capacità delle MAIT nei pazienti R di produrre IFN-g e GRZM-B, solo al T0. L'analisi in silico di dataset pubblici ha rivelato la presenza delle MAIT all'interno delle lesioni primarie e metastatiche, e dopo terapia il loro livello era aumentato nelle lesioni che regredivano. Infine, abbiamo correlato il livello mediano delle cellule MAIT circolanti (1,7% delle cellule T CD8) con la risposta alla terapia. Utilizzando questo valore come soglia, i pazienti che presentavano una percentuale di MAIT al di sopra della soglia hanno mostrato una risposta migliore alla terapia. L’analisi delle cellule T CD8 dei pazienti con mRCC ha rivelato la presenza di 61 clusters. A causa del numero basso di pazienti che rispondono alla terapia, abbiamo trovato percentuali simili tra i clusters dei pazienti R e NR sia prima che dopo terapia. Tuttavia, analizzando insieme tutti i pazienti trattati con anti-PD1 (n=19), si osserva come la terapia abbia indotto una ridistribuzione di diverse sottopopolazioni di cellule T CD8. In particolare, le cellule T staminali di memoria (TSCM) sono diminuite dopo il terzo ciclo di terapia. Al contrario, le sottopopolazioni di cellule EM sono aumentate in percentuale dopo la terapia. In termini di funzionalità, abbiamo osservato che, se comparate con le cellule EM, le TSCM dopo la terapia perdono la capacità di proliferare sebbene mantengano una più alta polifunzionalità, producendo simultaneamente TNF e IFN-g. In conclusione, abbiamo fornito l’evidenza che i pazienti mM e mRCC rispondono in modo diverso alla terapia con anti-PD1, i primi sono caratterizzati da più alti livelli di cellule MAIT attivate nei pazienti R, mentre i secondi sono caratterizzati da un pool probabilmente esaurito di TSCM circolante.CD8 T lymphocytes play a central role in immunity to cancer through their capacity to kill malignant cells. However, prolonged exposure to cognate antigens in tumor microenvironment contribute to induce severe CD8 T cell exhaustion (Tex). During the last decade cancer treatment has been revolutionized by immune check point inhibitors (ICIs) that block the activity of inhibitor receptors, such as PD-1 present on the surface of Tex cells, reinvigorating them. Despite observations of durable responses to ICI therapy, not all patients respond to the treatment. Therefore, we focused our attention on identifying alterations that occur in circulating CD8 T lymphocytes of metastatic melanoma (mM) and metastatic renal-cell carcinoma (mRCC) patients during treatment with anti-PD1, in order to understand how and why some patients respond or not to ICI therapy. The first cohort comprised 28 patients with mM, 17 of whom were defined responders (R) whereas 11 non-responders (NR). Cryopreserved PBMC, obtained prior to initiating therapy (T0) after the first (T1) and second therapy cycles (T2), was studied by 30 parameter high-dimensional flow cytometry in combination with single cell RNA-sequencing (scRNAsq). The second cohort comprised 19 patients with mRCC, 5 of which were defined responders (R) whereas 14 non-responders (NR). Cryopreserved PBMC, obtained prior to initiating therapy (T0) after the first (T1), second (T2) and third therapy cycle (T3), was studied by 28 flow cytometry and analyzed using FAUST, a novel non-parametric method for unsupervised discovery of cell population. In R patients we found an increase of proliferating effector memory (EM) cells expressing high level of Ki67, ICOS, CD95, HLA-DR, CD71, CD98, CXCR6, granulysin and CD38, both before and after first and second cycle of ICI therapy. scRNA-seq revealed the presence of activated mucosal associated invariant T (MAIT) cells expressing CD69 and CXCR4, and their increase in R compared to NR, before and after ICI therapy. Along with increased percentage of peripheral MAIT cells we also observed greater ability to produce IFN-g and GRZM-B in R patients, before starting therapy but not after. In silico analysis of public datasets revealed the presence of MAIT cells within primary and metastatic lesions and their increased level within lesions regressing after ICI. Finally, to associate our finding with clinical outcomes, we correlated the median level (1.7% of CD8 T cells) of circulating MAIT cells with the response to therapy. Using this value as a threshold, patients who exhibited MAIT frequency above the threshold showed a better response to therapy. Flow cytometry reveals that CD8 T cells from mRCC patients could be classified in 61 cell clusters. Due to the relatively low number of patients responding to therapy, we found similar percentages of clusters in R and NR at all timepoints. However, considering all patients treated with anti-PD1 (n=19), therapy induced a redistribution of different subpopulations of CD8 T cells. In particular, T stem cell memory (TSCM) decreased after the third cycle of therapy. On the contrary, the EM compartments increased after therapy. In terms of functionality, we observed that, if compared with EM, after therapy TSCM lost proliferative potential but retained more polyfunctionality, producing simultaneously TNF and IFN-g. In conclusion, we provide evidence that mM and mRCC patients differently respond to anti-PD1 therapy, the formers are characterized by more activated MAIT in R patients, while the latter are characterized by a pool of circulating likely-exhausted TSCM

Effective Treatment of Patients Experiencing Primary, Acute HIV Infection Decreases Exhausted/Activated CD4+ T Cells and CD8+ T Memory Stem Cells

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 (TSCM) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted TSCM. 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

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

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

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