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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., Competing Interests: Each contributing author declares to have no competing interests., (Copyright © 2020 Iole Macchia et al.)

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.

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.

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.

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 P-HIV 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.

Despite a long-lasting global effort, the Holy Grail quest for a protective vaccine, able to confer prevention to HIV infection, did not reach the hoped for results, nor seems able to do so in the near future. Since mucosal surfaces of the host serve as the main entry point for HIV, it seems now logical to switch from a systemic to a localized view of events, in order to reveal critical steps useful in designing new and different vaccination strategies. In this context, the recent description of the very early phases of infection, from the eclipse to the viremia peak phase, seems to define a point-of-no-return threshold after which the main HIV infection steps, i.e. the massive destruction of the CD4+CCR5+ cell pool, the destruction of the mucosal physical barrier, and the establishment of reservoir sanctuaries, have already been accomplished. Nevertheless, the underlying mechanisms, the timing, and the consequences of evasion mechanisms exploited by HIV are still under scrutiny. Innate immunity, as part of a rapid lymphoid stress surveillance system, is known to play a central role in host responses to many infectious agents. In particular, Vγ9Vδ2 T-cells are able to quickly respond to danger signals without the need for classical major histocompatibility complex presentation, and may act as a bridge between innate and acquired arms of immune response, being able to kill infected/transformed cells, release antimicrobial soluble factors, and increase the deployment of other innate and acquired responses. Many experimental evidences suggest a direct role of circulating Vγ9Vδ2 T-cells during HIV disease. They may exert a direct anti-HIV role by secreting chemokines competing for HIV entry coreceptors as well as other soluble antiviral factors, and by killing infected cells by cytotoxic natural killer-like mechanisms. Moreover, they were found progressively depleted and anergic in advanced stages of HIV disease, this effect being directly linked to uncontrolled HIV replication. Scarce evidences are available on the involvement of mucosal gamma/delta T-cells during the early phases of HIV infection. In particular, the relative cause/effect links between HIV infection, destruction of the mucosal physical barrier, nonspecific activation of the immune system, and mucosal innate cell activation and effector functions, are still not completely defined. In order to attain an effective manipulation of innate immune cells, aiming at the induction of an effective adaptive immunity against HIV, any information on the role of mucosal antiviral factors and innate immune cells will be very important. The aim of this review is to summarize the information on the role of gamma/delta T-cells during HIV infection, from the general circulating population to mucosal sites, in order to better describe areas deserving increased attention. In particular, strategies enhancing gamma/delta T-cell functions may open the possibility to formulate new immunotherapeutic regimens, which could impact the improvement of immune control of HIV disease.

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T(reg)), but it is also present on activated T cells, thus hindering correct T(reg) identification. Using classical markers for T(reg) recognition, discordant results were found in terms of T(reg) expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T(reg) detection. The objectives of this study were: (i) to identify T(reg) expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T(reg) are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T(reg) function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T(reg) were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39(+) cells within the CD4(+)CD25(high) cells have T(reg) properties (absence of interferon-gamma production and transforming growth factor-beta1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T(reg) by classical or novel markers. In contrast, a significantly higher percentage of T(reg) was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39(+) T(reg) increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T(reg) identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

The aim of the present study was to explain the observed anergy of Vgamma9Vdelta2 T cells from human immunodeficiency virus (HIV)-positive patients. CD3zeta expression and interferon (IFN)-gamma production by Vgamma9Vdelta2 T cells from HIV-positive and HIV-negative subjects were analyzed. We demonstrated that Vgamma9Vdelta2 T cells from HIV-infected patients expressed a lower level of CD3zeta than did Vgamma9Vdelta2 T cells from healthy donors. A direct correlation was found between CD3zeta expression and IFN-gamma production capability by Vgamma9Vdelta2 T cells. However, activation of protein kinase C by phorbol myristate acetate is able to restore CD3zeta expression and IFN-gamma production. Our findings may contribute to clarification of the molecular mechanisms of Vgamma9Vdelta2 T cell anergy found in HIV-positive patients.

Vgamma9Vdelta2 T cells display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. The activation of Vgamma9Vdelta2 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 Vgamma9Vdelta2 T cells to induce granulocyte activation. We showed that soluble factors released by ZOL-stimulated Vgamma9Vdelta2 T cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. Proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated Vgamma9Vdelta2 T cells. Moreover, MCP-2 depletion by neutralizing Ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. Altogether, these data show a Vgamma9Vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of Vgamma9Vdelta2 T-lymphocytes in orchestrating the immune response. In conclusion, an immune modulating strategy targeting Vgamma9Vdelta2 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.

Several signals influence dendritic cell (DC) functions and consequent the immune responses to infectious pathogens. Our recent findings provide a new model of intervention on DCs implicating human gammadelta T cell stimuli. Vgamma9Vdelta2 T cells represent the major subset of circulating human gammadelta T cells and can be activated by non-peptidic molecules derived from different microorganisms or abnormal metabolic routes. With activated-Vgamma9Vdelta2 T cell co-culture, immature DCs acquire features of mature DCs, such as increasing the migratory activity, up-regulating the chemokine receptors, and triggering the Th1 immune response. Similar to the NK-derived signals, DC activation is mediated by soluble factors as well as cell-to-cell contact. Many non-peptidic molecules including nitrogen-containing bisphosphonates and pyrophosphomonoester drugs, can stimulate the activity of Vgamma9Vdelta2 T cells in vitro and in vivo. The relatively low in vivo toxicity of many of these drugs makes possible novel vaccine and immune-based strategies against infectious diseases.

In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and gammadelta T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of Vgamma9Vdelta2 T cells (the major subset of gammadelta T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, Vgamma9Vdelta2 T cells increase both their expression of CD69 and CD25 and the production of TNF-alpha and IFN-gamma, in contrast to DC treated with Vgamma9Vdelta2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory Vgamma9Vdelta2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of Vgamma9Vdelta2 T cells in the control of mycobacterial infection.

Phosphoantigens are mycobacterial non-peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for gammadelta T cell responses to mycobacteria. Thus here, the immunogenicity of 6-kDa early secretory antigenic target-mycolyl transferase complex antigen 85B (ESAT-6-Ag85B) (H-1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime-boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL-2, IL-6, IFN-gamma, TNF-alpha), it further anergized blood gammadelta T lymphocytes selectively. By contrast, the hybrid H-1 induced only memory alphabeta T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN-gamma-secreting cells (+ 40 cells/million PBMC, equally specific for ESAT-6 and for Ag85B). Hence, in macaques, a prime-boost with the H-1/phosphoantigen subunit combination induces two waves of immune responses, successively by gammadelta T and alphabeta T lymphocytes.

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.

Natural T (NT) lymphocytes recognize infected cells or microbial compounds without the classical genetic restriction of polymorphic major histocompatibility complex (MHC) molecules. NT cells are mainly composed of alphabeta and gammadelta T lymphocytes that express natural killer (NK) receptors and recognize preferentially various nonpeptidic antigens. Similar to NK cells, NT lymphocytes can see and kill target cells deficient in the expression of one or more MHC class I molecules. NT cells expressing the alphabeta TCR can recognize lipid and lipoglycan antigens presented in the context of nonpolymorphic CD1 molecules, whereas phosphocarbohydrates and alkylamines induce constitutive response of Vgamma9Vdelta2 T cells. The stimulation of Vgamma9Vdelta2 T cells with phosphocarbohydrates induces the production of cytokines (IFNgamma and TNFalpha) and the release of chemokines with suppressive activity on HIV replication. In addition, stimulated Vgamma9Vdelta2 T cells exert a cytolytic activity against HIV-infected targets. In HIV-infected patients, a quantitative and qualitative alteration is observed early during the infection. Vgamma9Vdelta2 T cells are deleted and the remaining gammadelta cells are anergic. Th1 cytokines (IL-12 and IL-15) positively regulate cytokine production by Vgamma9Vdelta2 T cells but they are inefficient in restoring normal functions in patients' gammadelta T cells. Interestingly, partial restoration of the immune system under highly active antiretroviral therapies (HAART) is associated to the recovery of functional Vgamma9Vdelta2 T cells. A large panel of phosphocarbohydrates able to selectively stimulate Vgamma9Vdelta2 T cells is currently available, and preliminary experiments in monkeys suggest their in vivo efficacy in helping to control SIV replication. These observations prompt the question of new immune intervention involving molecules that stimulate NT cells.

In humans, the circulating pool of mycobacteria-reactive Vgamma9Vdelta2+ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of Vgamma9Vdelta2+ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated gammadelta T cells have lost CD27 expression. In contrast, the CD27-CD45RA-Vgamma9Vdelta2+ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating Vgamma9Vdelta2+ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN-gamma-producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting gammadelta T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.