Silvia Heltai

Autocrine Nitric Oxide Modulates CD95-induced Apoptosis in γδ T Lymphocytes

γδ T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by γδ T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

Mycobacterium tuberculosisexploits the CD95/CD95 ligand system of γ δ T cells to cause apoptosis

Vγ9/Vδ2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if γ δ T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature α β T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the γ δ TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+ /CD95L− γ δ T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95‐blocking antibodies. The transient accumulation at the site of infection of CD95L+ γ δ lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.

Engagement of CD30 shapes the secretion of cytokines by human γ δ T cells

CD30 is a member of the TNF receptor superfamily, previously shown to be expressed on Hodgkins lymphoma cells and on normal activated lymphocytes. We here show that CD30 is highly expressed on recently activated human γ δ T cells. Elevated surface levels of this molecule persisted in long‐term cultures of γ δ cells, without further cell stimulation. CD30 acted as a co‐stimulus in γ δ T cells by potentiating the intracellular Ca2+ fluxes induced by CD3 cross‐linking. The engagement of CD30 enhanced the expression of several cytokines induced upon CD3 stimulation such as IL‐4 and IFN‐γ but not IL‐10. The CC chemokines RANTES and macrophage inflammatory protein‐1β were constitutively expressed and not affected by stimulation. The inducible expression of the neutrophil chemoattractant IL‐8 was enhanced by CD30 co‐stimulation, as well as that of the CC chemokines I‐309 and MDC, whereas the secretion of the monocyte chemotactic protein‐1 was not detected. Triggering of CD30 may therefore modulate the expression of several cytokines released by γ δ cells; the expression of its physiologic ligand by APC and neutrophils at the site of infection may contribute to determine the outcome of an immune response.

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation

ABSTRACT Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.

In vivo administration of GM-CSF promotes the clearance of apoptotic cells: effects on monocytes and polymorphonuclear leukocytes.

The clearance of apoptotic cells is crucial to avoid chronic inflammation and autoimmunity. Little is known about the factors that regulate it in vivo. We show that granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) administration to carcinoma patients confers to their leukocytes a significantly higher ability to phagocytose apoptotic cells than before (P < 0.005). GM‐CSF increased the concentration of monocytes and polymorphonuclear leukocytes in the peripheral blood and activated circulating polymorphonuclear leukocytes. Both effects abated early after treatment, whereas phagocytosis of apoptotic cells was still significantly higher after 18 days compared with basal values (P < 0.005 and P < 0.025 for monocytes and polymorphonuclear leukocytes, respectively). On in vitro phagocytosis of apoptotic cells monocytes, but not polymorphonuclear leukocytes, up‐regulated MHC class II membrane expression. These findings are consistent with the possibility that GM‐CSF endows both scavenger and antigen‐presenting leukocytes with the ability to internalize apoptotic tumor cells. J. Leukoc. Biol. 67: 174–182; 2000.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

BackgroundT-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-γ-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level.ResultsThe cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-γ producing T-cells were also producing MIP-1β whereas T-cells characterized by the sole production of IFN-γ were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-γ+ MIP-1β+ T-cells was equivalent to the measurement of the total IFN-γ+ T-cells, we adopted the IFN-γ+ MIP-1β+ data analysis system to evaluate IFN-γ-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-γ+ MIP-1β+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay.ConclusionThe IFN-γ+ MIP-1β+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

Blockade of the Fas-triggered intracellular signaling pathway in human melanomas is circumvented by cytotoxic lymphocytes.

Fas and Fas ligand (FasL) have been found both in lymphoid and in non‐lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system. Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas‐induced apoptosis of human T lymphocytes clones. Conversely, cross‐linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase‐3 activation, pointing to an early alteration in the Fas‐triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism. This suggests that melanoma cells evade immune‐mediated Fas‐triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas‐induced death via granzyme‐mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer. Int. J. Cancer 81:573–579, 1999.

Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17−IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10− and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17−IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

Evidence for the involvement of phosphatidylinositol 3-kinase in fMLP-stimulated neutrophil adhesion to ICAM-1-transfected cells.

&NA; Phosphatidylinositol 3‐kinase (PI‐3K) controls important intracellular steps involved in inflammation, immunity, and cell growth. PI‐3K also modulates leukocyte integrin adhesiveness. In this study we evaluated the role of PI‐3K on neutrophil adhesion to intercellular adhesion molecule‐1 (ICAM‐1)‐transfected cells. N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐stimulated neutrophil adhesion was inhibited by wortmannin and LY294002, two unrelated PI‐3K inhibitors, whereas phorbol myristate acetate (PMA)‐induced neutrophil adhesion was not inhibited by them. After fMLP stimulation, a rapid activation of AKT and ERK was observed. However, only activation of AKT was reversed by the PI‐3K inhibitors. Neutrophil expression of the &bgr;2‐integrins Mac‐1, lymphocyte function‐associated antigen‐1(LFA‐1), and gp150.95 was not affected by wortmannin, nor was expression of the activation epitope recognized by MAB24. We conclude that (a) PI‐3K is involved in fMLP‐activated neutrophil adhesion to ICAM‐1‐transfected cells, (b) the mechanism involved is not mediated by the modulation of &bgr;2‐integrin expression or activation, and (c) another mechanism seems to involve the adhesion to ICAM‐1 when a cellular system of adhesion is used.

Nef-specific CD45RA+ CD8+ T cells secreting MIP-1β but not IFN-γ are associated with nonprogressive HIV-1 infection

BackgroundLong-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease.ResultsWe analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease.ConclusionThe novel antigen-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.