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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, V©9V™2 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 V©9V™2 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 onthe 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. © Permanyer Publications 2011.

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.

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.

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.

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.

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.

Several subsets of αβ regulatory T cells (Tregs) have been described and studied intensively, but the potential regulatory role of γδ T cells remains largely unclear. Lymphocytes expressing γδ TCR are involved in both innate and adaptive immune responses, and their major adult human peripheral blood subset (Vγ9Vδ2) displays a broad reactivity against microbial agents and tumors. In this study we report that γδ T lymphocytes with regulatory functions (Vδ2 Tregs) are induced in vitro in the presence of specific Ag stimulation and cytokines (TGF-β1 and IL-15). These cells express FOXP3 and, similarly as αβ Tregs, suppress the proliferation of anti-CD3/anti-CD28 stimulated-PBMC. Phenotypic and functional analyses of Vδ2 Tregs will very likely improve our understanding about the role of γδ T cells in the pathogenesis of autoimmune, infectious, and neoplastic diseases. Copyright © 2009 by The American Association of Immunologists, Inc.

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.