Search

The outcome of COVID-19 appears to be influenced by vitamin D status of population. Although epidemiological data indicate that COVID-19 produces more severe symptoms and higher mortality in elderly in comparison to young patients and in men in comparison to women to date sex and age differences in vitamin D status in infected patients have not been evaluated yet. In this study we evaluated the levels of circulating 25(OH)D in patients hospitalized for COVID-19 divided accordingly to their sex and age. We also correlated 25(OH)D levels with patient’s respiratory status (i.e., PaO2/FiO2 ratio) and with sex hormones plasma levels to analyze the potential relationship of these parameters. We found no significant differences in plasma levels of 25(OH)D between pre- and post-menopausal female patients and age matched male patients. Interestingly, the 25(OH)D plasma levels positively correlated to PaO2/FiO2 ratio only in young patients, regardless of their sex. We also found a significantly positive correlation between 17β-estradiol and 25(OH)D in elderly women and between testosterone and 25(OH)D in elderly men, supporting the role of sex hormones in maintaining 25(OH)D levels. In conclusion, we suggest that a synergy between vitamin D and sex hormones could contribute to the age-related outcome of COVID-19.

SARS-CoV-2 serum neutralization assay represents the gold standard for assessing antibody-mediated protection in naturally infected and vaccinated individuals. In the present study, 662 serum samples collected from February 2020 to January 2021 from acute and convalescent COVID-19 patients were tested to determine neutralizing antibody (NAb) titers using a microneutralization test (MNT) for live SARS-CoV-2. Moreover, anti-SARS-CoV-2 IgG, IgA, and IgM directed against different viral antigens were measured by high-throughput automated platforms. We observed higher levels of NAbs in elderly (&gt, 60 years old) individuals and in patients presenting acute respiratory distress syndrome. SARS-CoV-2 NAbs develop as soon as five days from symptom onset and, despite a decline after the second month, persist for over 11 months, showing variable dynamics. Through correlation and receiver operating characteristic (ROC) curve analysis, we set up a testing algorithm, suitable for the laboratory workload, by establishing an optimal cutoff value of anti-SARS-CoV-2 IgG for convalescent plasma donors to exclude from MNT samples foreseen to have low/negative NAb titers and ineligible for plasma donation. Overall, MNT, although cumbersome and not suitable for routine testing of large sample sizes, remains the reference tool for the assessment of antibody-mediated immunity after SARS-CoV-2 infection. Smart testing algorithms may optimize the laboratory workflow to monitor antibody-mediated protection in COVID-19 patients, plasma donors, and vaccinated individuals.

The immunological mechanisms underlying the clinical presentation of SARS-CoV-2 infection and those influencing the disease outcome remain to be defined. Myeloid-derived suppressor cells (MDSC) have been described to be highly increased during COVID-19, however, their role remains elusive. We performed an in depth analysis of MDSC in 128 SARS-CoV-2 infected patients. Polymorphonuclear (PMN)-MDSC expanded during COVID-19, in particular in patients who required intensive care treatments, and correlated with IL-1β, IL-6, IL-8, and TNF-α plasma levels. PMN-MDSC inhibited T-cells IFN-γ production upon SARS-CoV-2 peptides stimulation, through TGF-β- and iNOS-mediated mechanisms, possibly contrasting virus elimination. Accordingly, a multivariate regression analysis found a strong association between PMN-MDSC percentage and fatal outcome of the disease. The PMN-MDSC frequency was higher in non-survivors than survivors at the admission time, followed by a decreasing trend. Interestingly, this trend was associated with IL-6 increase in non-survivors but not in survivors. In conclusion, this study indicates PMN-MDSC as a novel factor in the pathogenesis of SARS-CoV2 infection, and open up to new therapeutic options.

Background Several biomarkers have been identified to predict the outcome of COVID-19 severity, but few data are available regarding sex differences in their predictive role. Aim of this study was to identify sex-specific biomarkers of severity and progression of acute respiratory distress syndrome (ARDS) in COVID-19. Methods Plasma levels of sex hormones (testosterone and 17β-estradiol), sex-hormone dependent circulating molecules (ACE2 and Angiotensin1-7) and other known biomarkers for COVID-19 severity were measured in male and female COVID-19 patients at admission to hospital. The association of plasma biomarker levels with ARDS severity at admission and with the occurrence of respiratory deterioration during hospitalization was analysed in aggregated and sex disaggregated form. Results Our data show that some biomarkers could be predictive both for males and female patients and others only for one sex. Angiotensin1-7 plasma levels and neutrophil count predicted the outcome of ARDS only in females, whereas testosterone plasma levels and lymphocytes counts only in males. Conclusions Sex is a biological variable affecting the choice of the correct biomarker that might predict worsening of COVID-19 to severe respiratory failure. The definition of sex specific biomarkers can be useful to alert patients to be safely discharged versus those who need respiratory monitoring.

Background/objectives A dysregulated inflammatory profile plays an important role in coronavirus disease-2019 (COVID-19) pathogenesis. Moreover, the depletion of lymphocytes is typically associated with an unfavourable disease course. We studied the role and impact of p53 and deacetylase Sirtuin 1 (SIRT1) on lymph-monocyte homeostasis and their possible effect on T and B cell signalling. Methods Gene expression analysis and flow cytometry were performed on peripheral blood mononuclear cells (PBMC) of 35 COVID-19 patients and 10 healthy donors (HD). Inflammatory cytokines, the frequency of Annexin+ cells among CD3+ T cells and CD19+ B cell subsets were quantified. Results PBMC from COVID-19 patients had a higher p53 expression, and higher concentrations of plasma proinflammatory cytokines (IL1β, TNF-α, IL8, and IL6) than HD. Deacetylase Sirtuin 1 (SIRT1) expression was significantly decreased in COVID-19 patients and was negatively correlated with p53 (p = 0.003 and r = −0.48). A lower expression of IL-7R and B Cell linker (BLNK), key genes for lymphocyte homeostasis and function, was observed in COVID-19 than in HD. The reduction of IgK and IgL chains was seen in lymphopenic COVID-19 patients. A significant increase in both apoptotic B and T cells were observed. Inflammatory cytokines correlated positively with p53 (IL-1β: r = 0.5 and p = 0.05; IL-8: r = 0.5 and p = 0.05) and negatively with SIRT1 (IL1-β: r = −0.5 and p = 0.04; TNF-α: r = −0.4 and p = 0.04). Conclusions Collectively, our data indicate that the inflammatory environment, the dysregulated p53/SIRT1 axis and low expression of IL7R and BLNK may impact cell survival, B cell signalling and antibody production in COVID-19 patients. Further studies are required to define the functional impact of low BLNK/IL7R expression during severe acute respiratory syndrome coronavirus-2 infection.

Tuberculosis (TB), due to Mycobacterium tuberculosis infection, is still the principal cause of death caused by a single infectious agent. The balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. Factors defining this variety are unclear and likely involve both mycobacterial and immunological components. Myeloid derived suppressor cells (MDSC) have been shown to be expanded during TB, but their role in human TB pathogenesis is not clear. We evaluated the frequency of circulating MDSC by flow-cytometry in 19 patients with active TB, 18 with latent TB infection (LTBI), and 12 healthy donors (HD) as control. Moreover, we investigated the capacity of MDSC to modulate the mycobactericidal activity of monocytes. The association between MDSC level and TB chest X-ray severity score was analyzed. We observed that, unlike active TB, polymorphonuclear (PMN)-MDSC are not expanded in LTBI patients, and, by performing a receiver operating characteristic (ROC) curve analysis, we found that PMN-MDSC frequency supported the discrimination between active disease and LTBI. Interestingly, we observed an association between PMN-MDSC levels and the severity of TB disease evaluated by chest X-ray. Specifically, PMN-MDSC frequency was higher in those classified with a low/mild severity score compared to those classified with a high severity score. Moreover, PMN-MDSC can impact mycobacterial growth by inducing ROS production in Bacillus Calmette et Guerin (BCG)-infected monocytes. This effect was lost when tested with M. tuberculosis (MTB), In conclusion, our data indicate that the elevated frequency of PMN-MDSC in IGRA-positive individuals is associated with active TB. Our findings also pointed out a beneficial role of PMN-MDSC during human active TB, most likely associated with the limitation of inflammation-induced tissue damage.

Increased production of inflammatory cytokines and myeloid-derived suppressor cells occurs in patients with coronavirus disease 2019. These inversely correlated with perforin-expressing natural killer (NK) and CD3+ T cells. We observed a lower number of perforin-expressing NK cells in intensive care unit (ICU) patients compared with non-ICU patients, suggesting an impairment of the immune cytotoxic arm as a pathogenic mechanism.

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

SARS-CoV-2 is associated with a 3.4% mortality rate in patients with severe disease. The pathogenesis of severe cases remains unknown. We performed an in-depth prospective analysis of immune and inflammation markers in two patients with severe COVID-19 disease from presentation to convalescence. Peripheral blood from 18 SARS-CoV-2-infected patients, 9 with severe and 9 with mild COVID-19 disease, was obtained at admission and analyzed for T-cell activation profile, myeloid-derived suppressor cells (MDSCs) and cytokine profiles. MDSC functionality was tested in vitro. In four severe and in four mild patients, a longitudinal analysis was performed daily from the day of admission to the early convalescent phase. Early after admission severe patients showed neutrophilia, lymphopenia, increase in effector T cells, a persisting higher expression of CD95 on T cells, higher serum concentration of IL-6 and TGF-β, and a cytotoxic profile of NK and T cells compared with mild patients, suggesting a highly engaged immune response. Massive expansion of MDSCs was observed, up to 90% of total circulating mononuclear cells in patients with severe disease, and up to 25% in the patients with mild disease; the frequency decreasing with recovery. MDSCs suppressed T-cell functions, dampening excessive immune response. MDSCs decline at convalescent phase was associated to a reduction in TGF-β and to an increase of inflammatory cytokines in plasma samples. Substantial expansion of suppressor cells is seen in patients with severe COVID-19. Further studies are required to define their roles in reducing the excessive activation/inflammation, protection, influencing disease progression, potential to serve as biomarkers of disease severity, and new targets for immune and host-directed therapeutic approaches.

Antiretroviral treatment (ART) of Primary HIV Infection (PHI) has demonstrated virological and immunological benefits. The effect of early ART during PHI on the level of growth factors and chemokines modulating immune cell functions remains to be established. The aim of our work was to analyze the dynamics of 27 cytokines, chemokines and growth/regulation factors in plasma of HIV infected patients treated during PHI. Patients with PHI (n = 43) were enrolled before, 24 and 48 weeks after therapy initiation. Quantification of soluble immune mediators was performed in plasma from HIV infected patients and healthy donors (HD, n = 7) by Luminex technology. The cytokines profile was strongly perturbed in primary HIV infected patients when compared to healthy donors (HD). After 48 weeks of ART, some of these factors were restored to HD level (IL-2, IL-5, IL-7, IL-9, IL12p70, TNFα) while others persisted higher than HD (IL-6, IL-10, IL-13). Interestingly, a subset of chemokines, such as IL-8, MCP-1, RANTES and CCL27, and growth factors such as HGF, SCF and GM-CSF, increased during ART, reaching values significantly higher than HD after 48 weeks. Moreover, the G-CSF and MIP-1β soluble mediators were persistently altered and showed an inverse correlation with the CD4/CD8 T cell ratio. The increase of chemokines with antiviral activity and of growth factors with hematopoietic and immunomodulatory properties may have beneficial effects. Other studies are mandatory to evaluate the effects of long lasting levels of these factors to clarify their possible role in the context of protection/pathogenesis.

The impact of early antiretroviral therapy (ART) during Primary HIV Infection (PHI) on the hematopoietic progenitor cells (HPCs) homeostasis is not available. This study aimed to characterize HPCs and their relationship with cytokines regulating progenitors function in ART-treated patients with PHI. We enrolled HIV infected patients treated with ART during PHI. Circulating HPCs, Lymphoid-HPCs (L-HPCs) frequency and plasmatic concentrations of IL-7, IL-18 and Stem Cell Factor (SCF) were analysed at baseline and after 6 months of therapy. ART introduction during PHI restored the decline of L-HPCs, induced a decrease in the level of pro-inflammatory IL-18 cytokine and a parallel increase of SCF. Moreover, L-HPCs frequency positively correlated with IL-18 at baseline, and with SCF after 6 months of therapy, suggesting that different signals impact L-HPCs expansion and maintenance before and after treatment. Finally, the SCF receptor expression on HPCs decreased after early ART initiation. These insights may open new perspectives for the evaluation of cytokine-driven L-HPCs expansion and their impact on the homeostasis of hematopoietic compartment during HIV 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.

The impact of HIV infection on the frequency and differentiation capability of CD34+ bone marrow hematopoietic progenitor cells (BM-HPCs) is still debated, having a possible primary role in antiretroviral-induced immunoreconstitution. We investigated the influence of HIV replication or proinflammatory cytokines on lymphopoietic capability of BM-HPCs from seven viremic (VR) and five nonviremic (NVR) HIV-infected patients. We found that BM-HPCs from VR patients were unable to differentiate in vitro toward T cells, and produced proinflammatory cytokines in the absence of viral replication. In contrast, the lymphoid differentiation potential of BM-HPCs was partially restored in successfully antiretroviral therapy-treated patients. We also showed that TLR8 triggering induced BM-HPCs from healthy donors to release proinflammatory cytokines affecting T cell differentiation. These data suggest that in HIV-infected patients, the lymphopoiesis capability of BM-HPCs may be modulated by a virus-driven autocrine mechanism involving proinflammatory cytokines.

Myeloid-derived suppressor cells (MDSC) are expanded during HIV-1 infection and correlated with disease progression. MDSC expand in the early phase of primary infection depending on TRAIL level. In this study we evaluated the effect of ART on the frequency of MDSC in patients with primary HIV infection (PHI), and their impact on CD4 T cell reconstitution. MDSC frequency was evaluated by flow-cytometry in 60 PHI patients at 12, 24 and 48 weeks after ART initiation. Cytokine plasma levels were evaluated by Luminex technology at the same time points. The capacity of MDSC to modulate hematopoietic early progenitor cells' expansion was evaluated using the OP9/Dl1 in vitro system. As previously described, polymorphonuclear-MDSC (PMN-MDSC) frequency was higher in PHI compared to healthy donors. Interestingly, 48 weeks of successful ART failed to normalize the PMN-MDSC frequency. Moreover, PMN-MDSC frequency was not correlated with residual viral load, suggesting that the persistence of PMN-MDSC was not due to residual viral replication. Interestingly, patients with low PMN-MDSC frequency (6%). We also found an inverse correlation between PMN-MDSC frequency and CD4-T cell count at 48 weeks post-ART, which was confirmed by multivariate analysis adjusting for age and CD4 T cell number at baseline. These data suggest that the persistence of PMN-MDSC may impact CD4 T cell recovery. Indeed, in vitro PMN-MDSC impaired the expansion of CD34+CD38- hematopoietic early progenitors. Further, a balance between TRAIL and GM-CSF may be necessary to maintain a low MDSC level. In conclusion, early ART initiation was not able to normalize PMN-MDSC frequency that might impact the CD4 T cell recovery. These data open new questions regarding the clinical impact of MDSC persistence in HIV+ patients, in particular on non-AIDS related diseases.

Primary and chronic human immunodeficiency virus (HIV) infection alters γδ T-cell features. However, there is no evidence about early combined antiretroviral therapy (cART) and γδ T-cell dynamics. In the present study, HIV-positive individuals were divided into those with early primary infection (EPI) and those with late primary infection (LPI). The analysis of γδ T cells was performed by flow cytometry before and after therapy. Polyfunctional profile was assessed after in vitro peripheral blood mononuclear cell (PBMC) exposure to specific antigens. The results show that primary infection induced an expansion of Vδ1 T cells in LPI. Before treatment, a massive activation of γδ T-cell subsets was observed in both groups of patients, that correlated with disease progression and was significantly reduced after cART introduction. Despite this, CD107A-expressing Vδ1 T cells in both groups were significantly fewer than in healthy donors, but were restored by therapy introduction. Polyfunctional analysis of Vδ1 T cells from HIV-positive individuals revealed a lower frequency of CD107A+CCL-4+ Vδ1 T-cell subsets than healthy donors that persists after therapy. Functional profile of Vδ2 was similar to that in healthy donors before therapy but, at 6months, a lower frequency of CD107A, interferon-γ- or tumor necrosis factor-α-producing Vδ2 T cells was observed in the EPI group. Finally, individuals with LPI showed a lower frequency of quadruple-functional Vδ2 T-cell subset. In conclusion, during primary HIV infection, the baseline Vδ1 T-cell activation is correlated with immune reconstitution potential. Moreover, an altered γδ polyfunctional profile occurred, persisting after cART. Further studies are needed to understand whether a longer treatment of primary infection may increase γδ T-cell functionality.

Myeloid derived suppressor cells (MDSC) is a heterogeneous subset of immature and mature cells of the myeloid lineage, undergoing expansion during pathologic conditions, and able to perform strong immune suppressive functions. It has been shown that cryopreservation selectively impacts the polimorphonuclear (PMN) MDSC viability and recovery, and alters the correct analysis of MDSC subsets. In laboratory practice, cryopreservation is often inevitable, in particular in multicenter studies where samples have to be shipped to a centralized laboratory. Aim of the present work was to set out a new protocol to evaluate the frequency of PMN-MDSC in thawed cells by flow-cytometry. PBMC were isolated from HIV+ patients and healthy donors, and were cryopreserved for at least ten days. After thawing, two different protocols were used: 1. standard protocol (SP) consisting of staining with the antibodies mix and then fixing with formalin 1%; 2. thawed protocol (TP) in which fixation foregoes the staining with the antibodies mix. Results showed that PMN-MDSC frequency in ex vivo PBMC evaluated by means TP was comparable to that analysed by SP, indicating that the protocol did not alter PMN-MDSC quantification in ex vivo cells. We then demonstrated that PMN-MDSC frequency in thawed PBMC tested by TP was almost identical to the frequency obtained in ex vivo cells evaluated by using SP. However, we observed that after three hours of culture post-thawing, PMN-MDSC were not assessable anymore with both SP and TP. In conclusion, we herein demonstrated that fixing PBMC soon after thawing and before antibody staining allows preservation of PMN-MDSC integrity and a reliable cells quantification. Thus, it is possible to phenotipically identify PMN-MDSC in cryopreserved PBMC, consenting adequate test precision and accuracy as well as making multicentre research more feasible.

γδ T cells represent less than 5% of circulating T cells; they exert a potent cytotoxic function against tumor or infected cells and secrete cytokines like conventional αβ T cells. As αβ T cells γδ T cells reside in the typical T cell compartments (the lymph nodes and spleen), but are more widely distributed in tissues throughout the body. For these reasons, some investigators are exploring the possibility of immunotherapies aimed to expand and activate Vδ2 T cells, or using them as Chimeric Antigen Receptor carriers. However, the role of immunosuppressive microenvironment on Vδ2 T cells during infections and cancers has not been completely elucidated. In particular, the effects of myeloid-derived suppressor cells (MDSC), largely expanded in such pathologies, were not explored. In the present work, we demonstrated that MDSC may inhibit IFN-γ production and degranulation of phosphoantigen-activated Vδ2 T cells. Moreover, the Vδ2 T cells cytotoxic activity against the Burkitt lymphoma cell line Daudi and Jurkat cell line were impaired by MDSC. The Arginase I seems to be involved in the impairment of Vδ2 T cell function induced by both tumor cells and MDSC. These data open a key issue in the context of Vδ2-targeted immunoteraphy, suggesting the need of combined strategies aimed to boost Vδ2 T cells circumventing tumor- and MDSC-induced Vδ2 T cells suppression.

Objective During HIV infection, a down-modulation of CD3ζ was found on T cells, contributing to T-cell anergy. In this work, we studied the correlation between myeloid-derived suppressor cells (MDSC) frequency and T-cell CD3ζ expression. Moreover, we investigated the mechanisms of CD3ζ decrease exploited by MDSC. Design and method CD3ζ expression and MDSC frequency were evaluated by flow cytometry on peripheral blood mononuclear cells from 105 HIV-positive (HIV+) patients. The role of MDSC in the modulation of the HIV-specific T-cell response was evaluated. The level of CD3ζ mRNA and ELF-1 protein were analysed by real-time-PCR and western blot, respectively. Results We found that granulocytic-MDSC (Gr-MDSC) were expanded in HIV+ patients compared with healthy donors; in particular, in cART-treated individuals a higher Gr-MDSC frequency was observed in patients with a CD4 T-cell count below 400 cells/μl. We found an inverse correlation between the percentage of Gr-MDSC and CD3ζ level. Moreover, in-vitro MDSC depletion induced the up-regulation of CD3ζ in T cells, restoring the functionality of αβ, but not γδ T cells. The in-vitro effect of isolated MDSC on CD3ζ expression was found cell contact-dependent, and was not mediated by previously described molecules. CD3ζ down-modulation corresponds to the decrease of its mRNA induced by silencing the transcription factor ELF-1. Conclusion Our data provide new knowledge on mechanisms used by Gr-MDSC in immune-modulation and on their role in the immune reconstitution during antiviral treatments.

Background Human Ebola infection is characterized by a paralysis of the immune system. A signature of αβ T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014–2015 occurred in West Africa, and to assess their association with the clinical outcome. Methodology/Principal findings Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. Conclusions/Significances Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells., Author summary Human Ebola infection presents a high lethality rate and is characterized by a paralysis of the immune response. The definition of the protective immune profile during Ebola infection represents a main challenge useful in vaccine and therapy design. In particular, the protective/pathogenetic involvement of innate immune cells during Ebola infection in humans remains to be clarified. Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Center in Guinea, and the profiling of innate immunity was correlated with the clinical outcome. Our results show that both effector Vδ2 T-cells and NK cells were associated with survival, suggesting their involvement in the complex network of protective response to EBOV infection.

First anti-HCV treatments, that include protease inhibitors in conjunction with IFN-α and Ribavirin, increase the sustained virological response (SVR) up to 80% in patients infected with HCV genotype 1. The effects of triple therapies on dendritic cell (DC) compartment have not been investigated. In this study we evaluated the effect of telaprevir-based triple therapy on DC phenotype and function, and their possible association with treatment outcome. HCV+ patients eligible for telaprevir-based therapy were enrolled, and circulating DC frequency, phenotype, and function were evaluated by flow-cytometry. The antiviral activity of plasmacytoid DC was also tested. In SVR patients, myeloid DC frequency transiently decreased, and returned to baseline level when telaprevir was stopped. Moreover, an up-regulation of CD80 and CD86 on mDC was observed in SVR patients as well as an improvement of IFN-α production by plasmacytoid DC, able to inhibit in vitro HCV replication.

Background It has been demonstrated that myeloid-derived suppressor cells (MDSC) are expanded in HIV-1-infected individuals and correlated with disease progression. The phase of HIV infection during which MDSC expansion occurs, and the mechanisms that regulate this expansion remain to be established. In this study, we evaluated the frequency of MDSC in patients during primary HIV infection (PHI) and factors involved in MDSC control. Methods Patients with PHI and chronic HIV infection (CHI) were enrolled. PHI staging was performed according to Fiebig classification, and circulating MDSC frequency and function were evaluated by flow cytometry. Cytokine levels were evaluated by Luminex technology. Results We found that granulocytic MDSC (Gr-MDSC) frequency was higher in patients with PHI compared with healthy donors, but lower than that in patients with CHI. Interestingly, Gr-MDSC expansion was observed in the early phases of HIV infection (Fiebig II/III), but it was not associated with HIV viral load and CD4 T-cell count. Interestingly, in PHI, Gr-MDSC frequency was inversely correlated with plasmatic level of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although a direct correlation was observed in CHI. Furthermore, lower level of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) was observed in PHI compared with that in CHI. In vitro experiments demonstrated that, differently from CHI, recombinant TRAIL-induced apoptosis of Gr-MDSC from PHI, an effect that can be abrogated by GM-CSF. Conclusion We found that Gr-MDSC are expanded early during PHI and may be regulated by TRAIL and GM-CSF levels. These findings shed light on the fine mechanisms regulating the immune system during HIV infection and open new perspectives for immune-based strategies.

The definition of the immunological response to Zika (ZIKV) infection in humans represents a key issue to identify protective profile useful for vaccine development and for pathogenesis studies. No data are available on the cellular immune response in the acute phase of human ZIKV infection, and its role in the protection and/or pathogenesis needs to be clarified. We studied and compared the phenotype and functionality of T-cells in patients with acute ZIKV and Dengue viral (DENV) infections. A significant activation of T-cells was observed during both ZIKV and DENV infections. ZIKV infection was characterized by a CD4 T cell differentiation toward effector cells and by a lower frequency of IFN-γ producing CD4 T cells. Moreover, a substantial expansion of CD3+CD4−CD8− T-cell subset expressing Vδ2 TCR was specifically observed in ZIKV patients. Vδ2 T cells presented a terminally differentiated profile, expressed granzyme B and maintained their ability to produce IFN-γ. These findings provide new knowledge on the immune response profile during self-limited infection that may help in vaccine efficacy definition, and in identifying possible immuno-pathogenetic mechanisms of severe infection.

Sir, ART effectively suppresses viral replication and controls infection for an undefined period of time; however, viral eradication is not achievable because of long-lived cellular HIV reservoirs. We previously showed that, in chronically infected subjects with undetectable plasma HIV-RNA, bone marrow CD34+ haematopoietic progenitor cells (HPCs) are apparently free of HIV replication, but are blunted in differentiation capability, and may harbour HIV-DNA even after a long period on successful ART. Moreover, in patients treated with successful ART for a very long time, a persistent impairment in the lymphopoietic capability of circulating CD34+ HPCs was found, and lymphopoiesis exhaustion resulted correlated to systemic immune activation, only partially reversed by prolonged ART. To date, the mechanisms of HIV-related lymphopoiesis dysfunction remain largely unexplained, and in particular, little information is available on the possibility of limiting the occurrence of irreversible damage by early ART introduction. We herein describe immune activation levels, T cell profile/response and circulating HPC kinetics in a patient with primary HIV infection receiving early treatment with ART. The patient was further followed for 12 months, and blood samples were analysed before (baseline) and after 2, 24 and 48 weeks of ART. A young adult male was recently diagnosed with HIV acute infection (Fiebig IV stage according to Fiebig et al.). Baseline plasma HIV-1 RNA was 1868262 copies/mL, and CD4+ T lymphocyte count was 389 cells/mm. Ritonavir-boosted darunavir, tenofovir+ emtricitabine and raltegravir were started on day 3 after diagnosis. After 12 weeks of ART, viral load dropped ,40 copies/mL and ART was simplified to rilpivirine+emtricitabine+tenofovir. Plasma HIV-RNA remained undetectable at all timepoints thereafter. The viro-immunological parameters are shown in Figure 1(a). CD4+ cell count steadily increased over time: 534, 1218 and 1072 cells/mL at weeks 2, 24 and 48, respectively. Proviral HIV-DNA, determined as described in Rozera et al., was 82479 copies/10 PBMC at baseline and 21534, 1752 and 6809 copies/10 PBMC at weeks 2, 24 and 48, respectively. CD8+ T cell activation, measured as CD38 expression by flow cytometry, paralleled plasma HIV-RNA viral load, reaching at week 24 the level found in healthy donors. On the other hand, the level of early CD8+ T cells, evaluated by CD127 expression, steadily increased from baseline to week 48 (Figure 1b). Peripheral blood CD4+ and CD8+ T cell differentiation was evaluated by CD45RA and CCR7 expression. As shown in Figure 1(c), the variation in CD4+ subsets included a decrease in effector memory (EM; CD45RA2/CCR72) and an increase in Research letters

The aim of the present study was to explain the observed anergy of Vγ9Vδ2 T cells from human immunodeficiency virus (HIV)-positive patients. CD3ζ expression and interferon (IFN)-γ production by Vγ9Vδ2 T cells from HIV-positive and HIV-negative subjects were analyzed. We demonstrated that Vγ9Vδ2 T cells from HIV-infected patients expressed a lower level of CD3ζ than did Vγ9Vδ2 T cells from healthy donors. A direct correlation was found between CD3ζ expression and IFN-γ production capability by Vγ9Vδ2 T cells. However, activation of protein kinase C by phorbol myristate acetate is able to restore CD3ζ expression and IFN-γ production. Our findings may contribute to clarification of the molecular mechanisms of Vγ9Vδ2 T cell anergy found in HIV-positive patients. © 2008 by the Infectious Diseases Society of America. All rights reserved.

Gut-associated immune system has been identified as a major battlefield during the early phases of HIV infection. γδ T-cells, deeply affected in number and function after HIV infection, are able to act as a first line of defence against invading pathogens by producing antiviral soluble factors and by killing infected cells. Despite the relevant role in mucosal immunity, few data are available on gut-associated γδ T-cells during HIV infection. Aim of this work was to evaluate how primary (P-HIV) and chronic (C-HIV) HIV infection affects differentiation profile and functionality of circulating and gut-associated Vδ1 and Vδ2 T-cells. In particular, circulating and mucosal cells were isolated from respectively whole blood and residual gut samples from HIV-infected subjects with primary and chronic infection and from healthy donors (HD).Differentiation profile and functionality were analyzed by multiparametric flow cytometry. P-HIV and C-HIV were characterized by an increase in the frequency of effector Vδ1-T cells both in circulating and mucosal compartments. Moreover, during PHIV mucosal Vδ1 T-cells expressed high levels of CD107a, suggesting a good effector cytotoxic capability of these cells in the early phase of infection that was lost in C-HIV. P-HIV induced an increase in circulating effector Vδ2 T-cells in comparison to C-HIV and HD. Notably, P-HIV as well as HD were characterized by the ability of mucosal Vδ2 T-cells to spontaneously produce IFN-γ that was lost in C-HIV. Altogether, our data showed for the first time a functional capability of mucosal Vδ1 and Vδ2 T-cells during P-HIV that was lost in C-HIV, suggesting exhaustion mechanisms induced by persistent stimulation. Copyright

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-1β in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 Tcellresponse 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. Copyright

Antiretroviral therapy allows a restoration of immune cell homeostasis associated with a normal immune competence. Our goal was to analyze the modulation of polyfunctional HIV-specific CD8+ T-cell responses during antiretroviral therapy. HIV-infected individuals were divided into four groups according to CD4+ cell count and viral load at the moment of recruitment. Whole blood was stimulated with a pool of CD8-specific HIV-antigens to assess cytokine/chemokine production and cytotoxicity activity by using flow cytometry. The groups show different modulation in HIV-specific CD8+ T-cell responses. In particular, immunological failure showed different distributions of polyfunctional HIV-specific CD8+ responses, mainly due to an increase of cells producing CD107a/IFNγ/IL-2/MIP-1β. Our results indicate that this particular 4+ functional subset is a possible correlate of immunological failure. Considering the complexity of interactions among HAART, immune system and HIV, work is in progress to find correlates of therapy efficacy. Copyright © by BIOLIFE, s.a.s.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Background Interferon-gamma (IFN-γ) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-γ response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT). Methods The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-γ whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-γ response (7-day) to RD1 proteins in diluted whole blood was performed. Results Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-γ levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%). Conclusion These results indicate that IFN-γ long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.

Vγ9Vδ2 T cells display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. The activation of Vγ9Vδ2 T cells by aminobisphosphonate drugs such as zoledronic acid (ZOL) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells. The aim of this work was to evaluate the ability of soluble factors released by ZOL-activated Vγ9Vδ2 T cells to induce granulocyte activation. We showed that soluble factors released by ZOL-stimulated Vγ9Vδ2 T cells activate granulocytes by inducing their chemotaxis, phagocytosis, and α-defensins release. Proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated Vγ9Vδ2 T cells. Moreover, MCP-2 depletion by neutralizing Ab revealed a critical role of this chemokine in induction of granulocyte α-defensins release. Altogether, these data show a Vγ9Vδ2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of Vγ9Vδ2 T-lymphocytes in orchestrating the immune response. In conclusion, an immune modulating strategy targeting Vγ9Vδ2 T cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. Copyright © 2008 by The American Association of Immunologists, Inc.

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.