Patrizia Rovere

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.

Delayed clearance of apoptotic lymphoma cells allows cross-presentation of intracellular antigens by mature dendritic cells

Single cells are deleted from the midst of living tissue during normal turnover and embryogenesis. This event is not associated with inflammation or autoimmunity. Little is known of the clearance of apoptotic cells during dangerous situations, accompanied by extensive cell death and tissue damage: when macrophages are overwhelmed by apoptotic cells, other phagocytes, including immature dendritic cells (DCs), may become involved. DCs efficiently present antigens derived from the processing of internalized apoptotic bodies to class I‐ and class II‐restricted T cells. Antigen presentation results either in T cell activation or in their functional blockade. The outcome is influenced by pro‐inflammatory maturative signals: efficient T cell cross‐priming requires fully mature DCs. Here we discuss in vitro data suggesting that the number of apoptotic cells that die at a given time influences DC maturation and therefore their ability to uptake antigens from apoptotic cells and cross‐activate T lymphocytes. J. Leukoc. Biol. 66: 345–349; 1999.

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.

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.

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.

Cytokine secretion associated with the clearance of apoptotic bodies in renal cell carcinoma patients

The factors determining the outcome of immunotherapy in metastatic renal cell carcinoma (RCC) patients remain elusive. Macrophages from normal donors that phagocytose apoptotic cells secrete the immunosuppressive cytokine IL‐10 in vitro. Conversely, IL‐10 genetic deletion enhances the immunogenicity of apoptotic tumor cells in vivo. Elevated pre‐treatment levels of IL‐10 are associated with an unfavorable outcome of RCC. We examined whether the ability to release IL‐10 by macrophages from RCC patients that phagocytosed apoptotic cells correlated with the outcome of immunotherapy. To this aim, we derived macrophages from 30 patients with metastatic RCC and from 21 healthy subjects (11 sex‐ and age‐matched healthy controls and 10 younger donors). Patients either had a clinical response after immunotherapy, with a median survival after treatment of more than 18 months (n = 16), or were beginning immunotherapy after diagnosis of metastatic disease (n = 14). Macrophages from responding patients challenged with apoptotic cells released significantly less IL‐10 than controls (p = 0.0075) and recently diagnosed patients (p = 0.0198), as ascertained by a 2‐sided Students t‐test. This was not because macrophages from responding patients lost the ability to secrete IL‐10, because antibody opsonization of apoptotic cells rescued IL‐10 secretion. In contrast, macrophages from all groups of donors released similar amounts of TNF‐α. The failure in IL‐10 secretion by engulfing macrophages of responding subjects may exalt the immunogenicity of dying tumor cells, contributing to the success of immunotherapy.