NKG2A inhibits NKG2C effector functions of γδ T cells: implications in health and disease.

The CD94/NKG2 complex is expressed on T and NK lymphocytes. CD94 molecules covalently associate to activating or inhibitory NKG2 molecules, and their expression finely tunes cell responses. Human γδ T cells express several NKRs. Expression of these receptors is confined to the cytolytic Vδ2 subset, which coexpresses the FcγRIII CD16 and CD45RA and has been defined as Vγ9Vδ2 T(EMRA) cells. We show that the CD94/NKG2C complex, associated with KARAP/DAP12, is fully functional in γδ T cells, as determined by measuring IFN-γ production, T cell proliferation, and cytolytic activity by γδ lymphocytes. In contrast, NKG2A expression was found on all γδ T cell memory subsets, suggesting a crucial role of the inhibitory signal provided by this receptor on γδ T cell responses. Moreover, we found Vγ9Vδ2 T(EMRA), NK, and CD8+ αß T cells coexpressing NKG2A and NKG2C receptors. Functional experiments showed that the inhibitory signal mediated by the NKG2A receptor prevails when double-positive cells are activated. Finally, NKG2A expression on γδ LDGL correlates with asymptomatic pathology, even in the presence of NKG2C coexpression, whereas in symptomatic patients affected by severe disease, the inhibitory NKG2A receptor is absent, and a variety of activatory NKRs was found. We propose that the silent behavior of γδ cells in LDGL patients is a result of effective inhibitory HLA class I receptors.

Thiopyrophosphoantigens: solid-phase synthesis and in vitro characterization of a new class of Vgamma9 Vdelta2 T cells activators.

The Vgamma9 Vdelta2 T cells mediate rapid, innate-like immune responses to pathogens and are important in several key immunoregulatory pathways, including those involved in infections and tumor development. Vgamma9 Vdelta2 T cells respond to low molecular weight isoprenoid phosphoantigens; the prototypic stimulatory compound is isopentenylpyrophosphate (IPP), an alkylphosphate intermediate of mevalonate metabolism that elicits proliferative, cytotoxic, and cytokine secretion responses. We studied the replacement of the pyrophosphate moiety with the thiopyrophosphate bioisostere, synthesizing thioanalogues of IPP and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP, the most potent natural antigen known to date). Once their in vitro efficacy and stability had been demonstrated, we synthesized a small library of compounds through the development of an innovative solid-phase strategy. Biological results confirmed thioHMBPP to be the best compound of this first series. Future aims are (i) the exploitation of the parallel solid-phase strategy to further explore structure-activity relationships of this new class of synthetic antigens and (ii) the determination of the PK/PD profile of thioHMBPP.

Zoledronic acid and interleukin-2 treatment improves immunocompetence in HIV-infected persons by activating Vgamma9Vdelta2 T cells.

OBJECTIVE: gammadelta T cells bearing the Vgamma9Vdelta2 T-cell receptor exert many antiviral effector functions in humans, including release of anti-HIV factors and direct cytotoxicity against virus-infected cells. Moreover, they are known to activate dendritic cells, improving antigen presentation function. After HIV infection, Vgamma9Vdelta2 T-cell number and reactivity are rapidly affected and they decrease upon disease progression. Bisphosphonate drugs such as zoledronic acid (Zol), used to treat bone diseases, have been shown to induce in vivo, in combination with interleukin-2, Vgamma9Vdelta2 T-cells' activation. The aim of this work was to verify whether the administration of Zol in combination with interleukin-2 in HIV-infected patients might improve Vgamma9Vdelta2 T-cell function, including immune adjuvancy mediated by gammadelta-dendritic cell cross-talk. DESIGN AND METHODS: In HIV patients naive to antiretroviral therapy, we analyzed the effect of combined Zol and interleukin-2 treatment, in comparison to Zol alone, on Vgamma9Vdelta2 T-cell number, maturation and function, on dendritic cell activation and on HIV-specific CD8 T-cell response. RESULTS: Zol and interleukin-2-combined treatment induced in-vivo Vgamma9Vdelta2 T-cell expansion and maturation. Paralleling Vgamma9Vdelta2 T-cell activation, increased dendritic cell maturation and HIV-specific CD8 T-cell responses were found. CONCLUSION: The specific modulation of Vgamma9Vdelta2 T-cell number and responsiveness after HIV infection may be at least transiently restored in vivo by Zol and interleukin-2 treatment. In this way, the immune effector mechanisms, secondary to Vgamma9Vdelta2 T-cell activation, were improved, suggesting a possible adjuvancy role of Zol and interleukin-2 treatment in restoring innate and specific competence in HIV-infected persons.

In vivo effects of zoledronic acid on peripheral gammadelta T lymphocytes in early breast cancer patients.

INTRODUCTION: Amino-bisphosphonates are potent activators of human gammadelta T cells. The aim of our study was to evaluate the immunomodulating properties of a single-dose of zoledronic acid (ZA) on gammadelta T cells in a select group of disease-free breast cancer patients with osteopenia. MATERIALS AND METHODS: Blood samples were obtained, from 23 patients, before and 7, 28, 56, 90 and 180 days after a single-dose (4 mg) of ZA and analyzed by flow cyometry. RESULTS: A significant decrease of the different gammadelta T cell subsets was observed: Naïve (CD3+/Vdelta2+/CD45RA+/CD27+) after 180 days (P < 0.01); Central Memory (CD3+/Vdelta2+/CD45RA-CD27+) after 28 (P < 0.05), 90 (P < 0.01) and 180 days (P < 0.01); and Effector Memory (CD3+/Vdelta2+/CD45RA-/CD27-) after 56 (P < 0.01) and 90 (P < 0.05) days. Based on the observed gammadelta T cells kinetics patients could be divided in two groups: "responders" that showed a significant decrease in total numbers of gammadelta T cells and "non-responders" that showed no significant change. However, in vitro phosphoantigen stimulation of patients cells did not show significant differences in terms of IFN-gamma response by Vdelta2 T cells. CONCLUSION: We describe for the first time a long-lasting activation of effector subsets of gammadelta T cells in disease-free breast cancer patients after a single-dose of ZA. Our results highlight the need to further investigate the clinical significance of the immunomodulating properties of N-BPs.

Cross-subtype immunity against avian influenza in persons recently vaccinated for influenza.

Avian influenza virus (H5N1) can be transmitted to humans, resulting in a severe or fatal disease. The aim of this study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for seasonal influenza A (H1N1)/(H3N2). A small frequency of CD4 T cells specific for subtype H5N1 was detected in several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T cells. We also observed that seasonal vaccination is able to raise neutralizing immunity against influenza (H5N1) in a large number of donors. No correlation between influenza-specific CD4 T cells and humoral responses was observed. N1 may possibly be a target for both cellular and humoral cross-type immunity, but additional experiments are needed to clarify this point. These findings highlight the possibility of boosting cross-type cellular and humoral immunity against highly pathogenic avian influenza A virus subtype H5N1 by seasonal influenza vaccination.

Gamma delta T cells and dendritic cells: close partners and biological adjuvants for new therapies.

The knowledge of several signals influencing Dendritic Cell (DC) functions is crucial to manipulate the immune system for new vaccination therapies. Our recent findings provide a new model of intervention on DC system suggesting novel therapeutic implications. T, NK, and gammadelta T cell stimuli may enhance DC maturation, Th polarization and trigger the adaptive immune response. Regulatory effects of gammadelta T cells on inflammation and immune responses may be mediated by their interaction with DCs and they are analyzed in the last years in humans and mice. In humans, Vgamma9Vdelta2 T cells represent the most part of circulating gammadelta T cells and are activated by non-peptidic molecules derived from different microorganisms or abnormal metabolic routes. They share both NK-like and effector/memory T cell features, and among these the possibility to interact with DCs. Co-culture of immature DCs with activated Vgamma9Vdelta2 T cells allows DCs to acquire features of mature DCs complementing the migratory activity, up-regulating the chemokine receptors, and antigen presentation. Similarly to the NK-derived signals, DC activation is mostly mediated by soluble factors secreted by gammadelta T cells. Many non-peptidic molecules including nitrogen-containing bisphosphonates and pyrophosphomonoester drugs 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, through DCs, for infectious and neoplastic diseases.

Central memory Vgamma9Vdelta2 T lymphocytes primed and expanded by bacillus Calmette-Guérin-infected dendritic cells kill mycobacterial-infected monocytes.

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.

Differential expression of Werner and Bloom syndrome genes in the peripheral blood of HIV-1 infected patients.

Human immunodeficiency virus (HIV)-induced immunodeficiency and immune-system aging share some analogies. Since Werner (WRN) and Bloom (BLM) helicases are crucial in cell repair and aging, their peripheral blood mononuclear cells (PBMC) mRNA levels were compared in HIV-1 infected patients and in normal donors. The mean levels of WRN mRNA were 3.7-fold higher in PBMCs from HIV-1 infected individuals in comparison to healthy donors, whereas BLM mRNA mean levels were slightly higher, although not significantly. WRN increase was positively correlated to CD4 and CD8 T-cell numbers, and also the percentage of naive T lymphocytes, and was observed also in T-cell subsets. Interestingly, a general trend toward increased WRN mRNA levels in individuals with lower viral load was observed, without association with patient age, time of seroconversion, and on/off antiretroviral therapy regimen. On the whole, this study shows that WRN and BLM are differentially modulated in HIV infection, as WRN--but not BLM--is significantly increased, suggesting that mechanisms different from defect or loss of helicase function, observed in WRN and BLM syndromes, may be at the basis of T-cell aging in HIV infection.

A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct gammadelta and alphabeta T cell responses in primates.

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.

Isoniazid prophylaxis differently modulates T-cell responses to RD1-epitopes in contacts recently exposed to Mycobacterium tuberculosis: a pilot study.

RATIONALE: Existing data on the effect of treatment of latent tuberculosis infection (LTBI) on T-cell responses to Mycobacterium tuberculosis (MTB)-specific antigens are contradictory. Differences in technical aspects of the assays used to detect this response and populations studied might explain some of these discrepancies. In an attempt to find surrogate markers of the effect of LTBI treatment, it would be important to determine whether, among contacts of patients with contagious tuberculosis, therapy for LTBI could cause changes in MTB-specific immune responses to a variety of RD1-antigens. METHODS AND RESULTS: In a longitudinal study, 44 tuberculin skin test+ recent contacts were followed over a 6-month period and divided according to previous exposure to MTB and LTBI treatment. The following tests which evaluate IFN-gamma responses to RD1 antigens were performed: QuantiFERON TB Gold, RD1 intact protein- and selected peptide-based assays. Among the 24 contacts without previous exposure that completed therapy, we showed a significant decrease of IFN-gamma response in all tests employed. The response to RD1 selected peptides was found to be more markedly decreased compared to that to other RD1 antigens. Conversely, no significant changes in the response to RD1 reagents were found in 9 treated subjects with a known previous exposure to MTB and in 11 untreated controls. CONCLUSION: These data suggest that the effect of INH prophylaxis on RD1-specific T-cell responses may be different based on the population of subjects enrolled (recent infection versus re-infection) and, to a minor extent, on the reagents used.

Enhancement of BCG-induced Th1 immune response through Vgamma9Vdelta2 T cell activation with non-peptidic drugs.

Since drug-activated gammadelta T cells promote dendritic cell (DC) maturation, we analyzed the effect of combining gammadelta T cell specific drugs with BCG in vitro. BCG-induced DC maturation was increased by bromohydrin-pirophosphate (BrHPP) or zoledronate (Zol)-activated gammadelta T cells. Specifically, the co-culture with activated Vgamma9Vdelta2 T cells with BCG-infected DC resulted in a significant increase of the expression of CD80, CD86, CD40 and CD25 molecules on DC. Moreover, DC were able to produce increased levels of TNF-alpha and synthesize ex novo IL-15 without altering the IL-10/IL-12 immunoregulatory pathway. Finally, the Th1 immunity induced by BCG-infected DC on naïve CD4 T cells was increased by gammadelta T cell activation with BrHpp or Zol. These data indicate that gammadelta T cell triggering drugs could be used to enhance the BCG induced Th1 immunity.

CXCR5 identifies a subset of Vgamma9Vdelta2 T cells which secrete IL-4 and IL-10 and help B cells for antibody production.

Vgamma9Vdelta2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microorganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of CXCR5 identifies a unique subset of Vgamma9Vdelta2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2, IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5+ Vgamma9Vdelta2 T cells as a distinct memory T cell subset with B cell helper function.

Region of difference 1 antigen-specific CD4+ memory T cells correlate with a favorable outcome of tuberculosis.

BACKGROUND: Interferon (IFN)-gamma response to region of difference (RD) 1 proteins (culture filtrate 10 and early secreted antigenic target 6) or overlapping peptides is a novel diagnostic marker of tuberculosis (TB) infection. Because we have recently shown that the response to certain peptides selected from RD1 allows discrimination between active TB (A-TB) and successfully treated TB (T-TB), we analyzed here the effector memory T cell profile and RD1-specific responses under the same clinical conditions. METHODS: T cell responses to RD1 antigens were analyzed in patients with either severe or mild A-TB (classified on the basis of radiological lesions) and in 2 sets of healthy control subjects--those who had been successfully treated (the T-TB control subjects) and those whose tuberculin skin test (TST) results were negative (the TST-negative control subjects). IFN-gamma -producing CD4+ effector T cells were monitored by flow-cytometric analysis and ex vivo enzyme-linked immunospot (ELISPOT) assay, whereas a "cultured" ELISPOT assay was used to determine the frequency of memory T cells. RESULTS: In the patients with severe A-TB, both CD4-mediated effector memory and central memory responses to the selected RD1 peptides were almost absent, whereas these responses were found in the majority of the patients with mild A-TB. In contrast, recognition of the selected RD1 peptides was detected in the T-TB control subjects only by expanding the central memory T cell pool. CONCLUSIONS: These data suggest a protective role for RD1 peptide-specific CD4+ effector T cells, which undergo clonal expansion during Mycobacterium tuberculosis replication and then a contraction phase after disease resolution, culminating in the generation of CD4+ memory T cells.

Anti-severe acute respiratory syndrome coronavirus immune responses: the role played by V gamma 9V delta 2 T cells.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV) strain. Analyses of T cell repertoires in health care workers who survived SARS-CoV infection during the 2003 outbreak revealed that their effector memory V gamma 9V delta 2 T cell populations were selectively expanded ~3 months after the onset of disease. No such expansion of their alpha beta T cell pools was detected. The expansion of the V gamma 9V delta 2 T cell population was associated with higher anti-SARS-CoV immunoglobulin G titers. In addition, in vitro experiments demonstrated that stimulated V gamma 9V delta 2 T cells display an interferon- gamma -dependent anti-SARS-CoV activity and are able to directly kill SARS-CoV-infected target cells. These findings are compatible with the possibility that V gamma 9V delta 2 T cells play a protective role during SARS.

Accumulation of dysfunctional effector CD8+ T cells in the liver of patients with chronic HCV infection.

BACKGROUND/AIMS: Hepatitis C virus (HCV) causes a chronic infection that can lead to fibrosis and carcinoma. Immune responses mediated by cytotoxic T lymphocytes (CTLs) could be involved in viral clearance or persistence, and therefore in determining the course of the disease. METHODS: Intrahepatic and peripheral blood CD8+T cells were obtained from 32 HCV-chronically infected patients and analysed by flow-cytometry for surface markers of differentiation, IFNgamma and TNFalpha production, degranulation capacity and perforin content, after CD3 triggering. Results were compared with those obtained from 13 patients with a non-viral liver disease. RESULTS: Intrahepatic CD8+T cells of HCV-infected patients, despite their phenotype of pre-terminally and terminally differentiated effectors (CCR7-CD45RA-/+), are poorly responsive to T cell receptor (TCR)-mediated stimulation compared with those obtained from uninfected subjects. This defect correlates with the severity of fibrosis, is more pronounced in patients with ALT<1.5xN than with ALT>1.5xNU/ml, and is not evident after mitogen stimulation. CONCLUSIONS: The present study describes the accumulation of hypo-responsive CD8+T cells in the liver of patients with chronic HCV infection. Understanding the mechanisms underlying this impairment may be helpful in the design of innovative strategies for HCV treatment.

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication.

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Vgamma9Vdelta2 T cell-mediated non-cytolytic antiviral mechanisms and their potential for cell-based therapy.

In healthy adult Homo sapiens, the most frequent circulating gammadelta T cells (Vgamma9Vdelta2) respond to pyrophosphomonoesters, alkylamines (together referred to as non-peptidic antigens, NpAgs) and nitrogen-containing bisphosphonates. The seemingly very low toxicity of bisphosphonate and pyrophosphomonoester drugs in vivo, may allow novel approaches to the immunotherapy of viral infections. For example, these drugs can be used to stimulate Vgamma9Vdelta2 T cells to release antiviral molecules that directly suppress virus replication without destroying the virus-replicating cells. In addition, the soluble molecules released by gammadelta T cells could boost the antiviral potency of cytotoxic T lymphocytes (CTLs) and promote antigen presentation. The relative therapeutic value of drug-induced direct antiviral and immunoregulatory activities may depend on the infecting virus as well as on the nature of protective immune responses.