CRUSTY: a versatile web platform for the rapid analysis and visualization of high-dimensional flow cytometry data.

Flow cytometry (FCM) can investigate dozens of parameters from millions of cells and hundreds of specimens in a short time and at a reasonable cost, but the amount of data that is generated is considerable. Computational approaches are useful to identify novel subpopulations and molecular biomarkers, but generally require deep expertize in bioinformatics and the use of different platforms. To overcome these limitations, we introduce CRUSTY, an interactive, user-friendly webtool incorporating the most popular algorithms for FCM data analysis, and capable of visualizing graphical and tabular results and automatically generating publication-quality figures within minutes. CRUSTY also hosts an interactive interface for the exploration of results in real time. Thus, CRUSTY enables a large number of users to mine complex datasets and reduce the time required for data exploration and interpretation. CRUSTY is accessible at https://crusty.humanitas.it/ .

Health impact of natural gas emission at Cava dei Selci residential zone (metropolitan city of Rome, Italy).

Natural gas hazard was assessed at Cava dei Selci, a residential neighbourhood of Marino (Rome) by a joint study of gas emissions and related health problems. Here a densely urbanized zone with 4000 residents surrounds a dangerous natural gas discharge where, along the years, dozens of animals were killed by the gas. Gas originates from Colli Albani volcano and consists mostly of CO2 with ~ 1 vol% of H2S. In recent years, several gas-related accidents occurred in the urbanized zone (gas blowouts and road collapses). Some houses were evacuated because of hazardous indoor air gas concentration. Gas hazard was assessed by soil CO2 flux and concentration surveys and indoor and outdoor air CO2 and H2S concentration measurements. Open fields and house gardens release a high quantity of CO2 (32.23 tonnes * day-1). Inside most houses, CO2 air concentration exceeds 0.1 vol%, the acceptable long-term exposure range. In several houses both CO2 and H2S exceed the IDLH level (Immediately Dangerous to Life and Health). An epidemiological cohort study was carried out on the residents of two Cava dei Selci zones with high (zone A) and medium (zone B) gas hazard exposure, using the rest of Marino as reference zone. We found excess mortality and emergency room visits (ERV) related to high exposure to CO2 and H2S; in particular, an increased risk of mortality and ERV for diseases of central nervous system (HR 1.57, 95% CI 0.76-3.25 and HR 5.82, 95% CI 1.27-26.56, respectively) was found among men living in zone A.

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

BACKGROUND & AIMS: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). METHODS: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. RESULTS: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. CONCLUSIONS: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. LAY SUMMARY: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

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.

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

CXCR3 Identifies Human Naive CD8+ T Cells with Enhanced Effector Differentiation Potential.

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Using a multi-method approach based on soil radon deficit, resistivity, and induced polarization measurements to monitor non-aqueous phase liquid contamination in two study areas in Italy and India.

Geochemical and geophysical surveys employing radon deficit, resistivity, and induced polarization (IP) measurements were undertaken on soil contaminated with non-aqueous phase liquids (NAPLs) in two different sites in India and in Italy. Radon deficit, validated through the comparison with average soil radon in reference unpolluted areas, shows the extension of contamination in the upper part of the unsaturated aquifers. In site 1 (Italy), the spill is not recent. A residual film of kerosene covers soil grains, inhibiting their chargeability and reducing electrical resistivity difference with background unpolluted areas. No correlation between the two parameters is observed. Soil volatile organic compounds (VOCs) concentration is not linked with radon deficit, supporting the old age of the spillage. NAPL pollution in sites 2a and 2b (India) is more recent and probably still active, as demonstrated by higher values of electrical resistivity. A good correlation with IP values suggests that NAPL is still distributed as droplets or as a continuous phase in the pores, strengthening the scenario of a fresh spill or leakage. Residual fraction of gasoline in the pore space of sites 2a and 2b is respectively 1.5 and 11.8 kg per cubic meter of terrain. This estimation is referred to the shallower portion of the unsaturated aquifer. Electrical resistivity is still very high indicating that the gasoline has not been strongly degraded yet. Temperature and soil water content influence differently radon deficit in the three areas, reducing soil radon concentration and partly masking the deficit in sites 2a and 2b.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

Human T memory stem (TSCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8+ T-cell differentiation and allows the generation of CD45RO- CD45RA+ CCR7+ CD27+ CD95+ -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human TSCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy.

HIV-Specific CD8 T Cells Producing CCL-4 Are Associated With Worse Immune Reconstitution During Chronic Infection.

BACKGROUND: Immunological nonresponse represents the Achilles heel in the combination antiretroviral therapy (cART) effectiveness, and increases risk of clinical events and death. CD8 T cells play a crucial role in controlling HIV replication, and polyfunctional HIV-specific CD8 T cells have been associated with nonprogressive HIV infection. However, the possible role of polyfunctional CD8 T cells in predicting posttreatment immune reconstitution has not yet been explored. The aim of this study was to identify functional markers predictive of immunological response to cART in chronic HIV-infected patients. METHODS: A cohort of chronic HIV-infected individuals naive to cART were enrolled in the ALPHA study. CD4/CD8 T-cell subsets, their differentiation/activation, as well as susceptibility to apoptosis were analyzed before and after 12 months of cART. Moreover, CD8 T cells polyfunctional response after HIV antigenic stimulation was also assessed. RESULTS: Results showed a significant correlation between worse CD4 T-cell restoration and low frequency of naive CD4 T cells, high frequency of effector memory CD4 T cells, and high susceptibility to apoptosis of CD4 T cells all before cART. Moreover, CD8 functional subsets expressing total C-C motif chemokine ligand 4 (CCL-4) or in combination with CD107a and interferon gamma (IFNγ) were negatively associated with immune reconstitution. CONCLUSIONS: In conclusion, our study shows that a more differentiated phenotype of CD4 T cells and CCL-4-producing CD8 T cells could represent valuable predictors of worse immune reconstitution. These parameters may be used as tools for identifying patients at risk of immunological failure during cART and eventually represent the basis for innovative therapeutic strategies.

Soil radon survey to assess NAPL contamination from an ancient spill. Do kerosene vapors affect radon partition ?

A soil radon-deficit survey was carried out in a site polluted with kerosene (Rome, Italy) in winter 2016 to assess the contamination due to the NAPL residual component in the vadose zone and to investigate the role of the vapor plume. Radon is indeed more soluble in the residual NAPL than in air or water, but laboratory experiments demonstrated that it is also preferentially partitioned in the NAPL vapors that transport it and may influence soil radon distribution patterns. Specific experimental configurations were designed and applied to a 31-station grid to test this hypothesis; two RAD7 radon monitors were placed in-series and connected to the top of a hollow probe driven up to 80-cm depth; the first instrument was directly attached to the probe and received humid soil gas, which was counted and then conveyed to the second monitor through a desiccant (drierite) cylinder capturing moisture and eventually the NAPL volatile component plus the radon dissolved in vapors. The values from the two instruments were cross-calibrated through specifically designed laboratory experiments and compared. The results are in agreement within the error range, so the presence of significant NAPL vapors, eventually absorbed by drierite, was ruled out. This is in agreement with low concentrations of soil VOCs. Accordingly, the radon-deficit is ascribed to the residual NAPL in the soil pores, as shown very well also by the obtained maps. Preferential areas of radon-deficit were recognised, as in previous surveys. An average estimate of 21 L (17 Kg) of residual NAPL per cubic meter of terrain is provided on the basis of original calculations, developed from published equations. A comparison with direct determination of total hydrocarbon concentration (23 kg per cubic meter of terrain) is provided.

Laboratory simulation of recent NAPL spills to investigate radon partition among NAPL vapours and soil air.

Soil radon is employed to trace residual NAPL (Non-Aqueous Phase Liquid) contamination because it is very soluble in these substances and is strongly depleted over polluted volumes of the subsoil. The solubility of radon into NAPL vapors, generally poorly considered, is investigated here, either as growth of radon exhalation from a material contaminated with increasing volumes of kerosene, or as radon partition between liquid kerosene, water and total air, considered ad the sum of kerosene vapors plus air.

Vγ9Vδ2 T-Cell Polyfunctionality Is Differently Modulated in HAART-Treated HIV Patients according to CD4 T-Cell Count.

Alteration of γδ T-cell distribution and function in peripheral blood is among the earliest defects during HIV-infection. We asked whether the polyfunctional response could also be affected, and how this impairment could be associated to CD4 T-cell count. To this aim, we performed a cross-sectional study on HIV-infected individuals. In order to evaluate the polyfunctional-Vγ9Vδ2 T-cell response after phosphoantigen-stimulation, we assessed the cytokine/chemokine production and cytotoxicity by flow-cytometry in HAART-treated-HIV+ persons and healthy-donors. During HIV-infection Vγ9Vδ2-polyfunctional response quality is affected, since several Vγ9Vδ2 T-cell subsets resulted significantly lower in HIV+ patients in respect to healthy donors. Interestingly, we found a weak positive correlation between Vγ9Vδ2 T-cell-response and CD4 T-cell counts. By dividing the HIV+ patients according to CD4 T-cell count, we found that Low-CD4 patients expressed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1ß in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 T-cell-response quality in Low-CD4 patients is specifically affected, suggesting a direct link between innate Vγ9Vδ2 T-cells and CD4 T-cell count. These findings suggest that Vγ9Vδ2 T-cell quality may be indirectly influenced by HAART therapy and could be included in a new therapeutical strategy which would perform an important role in fighting HIV infection.