Characterization of cord-blood derived gamma delta T cells immune response after stimulation with aminobisphosphonate compounds : their hypothetical role in the control of neonatal viral infections transmitted during pregnancy

Abstract

The innate immunity is the first defence line against microbes and pathogens, and exerts its protective activity by killing the infected host-cells, inducing an effective adaptive immunity. In microbial infections, adaptive and innate immune reactions cooperate to protect the host and, whenever it is possible, to eradicate or control the infection. Among its cellular components are mainly polymorphonuclear phagocytes, mononuclear phagocytes, Natural Killer (NK) cells, characterized by natural cytotoxic activity, and a minority population of T lymphocytes, that exhibit T cell receptor. T lymphocytes shown an highreactivity against bacteria, protozoa and viruses, and their percentage peripheral blood was significantly higher than normal in individuals with infections such as tuberculosis, salmonellosis, listeriosis, brucellosis, leishmaniasis, malaria, toxoplasmosis, infection by viruses such as Herpes Simplex (HSV-1)[Born WK et al. 2006; Roger Sciammas, Jeffrey A Bluestone. 1999]. The neonatal HSV diseases are often the result of infections developed in the last quarter of pregnancy with a risk of transmission between 30% and 60%. The immune system of the newborn in comparison with the adult is functionally immature, and it is different quantitatively and phenotypicaly in host defence [Bradley MB and Cairo MS 2005]. T cells isolated from cord blood (CB) compared to adult peripheral blood cells, show a reduced production of cytokines, a poor response to mitogenic stimulation, and reduced cytotoxic activity [Cairo C et al. 2008]. The immaturity of the neonatal immune system has been associated with increased susceptibility to various pathogens, resulting in higher mortality [Paula AV et al. 2006]. For this reason, aim of our project is to investigate the possibility to immunomodulate the innate immunity response as new therapeutical approach of neonatal pathologies. In cord blood T cells, which represent less than 1% of T lymphocytes, recognize non-peptide phospho-antigens expressed in microbial pathogens. These antigens were identified in bioactive fractions of M. tuberculosis and belong to isoprenoid pyrophosphates, a class of Pyrophosphomonoesters, such as isopentenil-pyrophosphate (IPP). Also synthetic molecules, known as Aminobisphosphonates (ABs), have been also identified as cell antigens. We have demonstrated that ABs, such as Pamidronate (PAM) and Zoledronate (ZOL), are able of stimulating T lymphocytes isolated from CBand differentiate into a regulatory phenotype. Initially we found that T cells isolated from CB respond significantly in terms of expansion to stimulation with PAM and ZOL, while stimulation with IPP does not involve any expansion. The response to these compounds is demonstrated by the membrane expression of activation markers such as CD25 and CD69, which are more present after stimulation. We also observed that treatments with PAM and ZOL induce the expansion of an effector population of T cells (CD62L-/CD27-) with regulatory phenotype (CD45RA-/CD16-), confirmed by their ability to produce specific cytokines (IFN and TNF). By flow cytometric analysis we observed that IL-2 or IL-2+ABs treatment induced V2+ T cells to differentiate into a non-cytotoxic phenotype that was confirmed by absence of intracellular perforin expression [Placido R et al, in press]. On the other hand, IL-2 or IL-2+ABs stimulation reduced significantly the non-cytotoxic phenotype of CB V2- T cells, suggesting their probable differentiation toward a cytotoxic subset. To characterize the CB cell-mediated cytotoxicity, we used cytotoxicity assay in which effector cells (CB) were incubated with targets cells (K562, human erythromyeloblastoid leukemia cell line) and cell death was evaluated at 6h after co-culture. IL-2 treated cells induced a significantly increase of tumor cell death (apoptosis). Since T lymphocytes are not able to acquire a cytotoxic phenotype and only IL2 stimulation induced cytotoxicity activity of CB cells, it was plausible that other cellular subsets could be involved in their cytolytic activity, as Natural Killer (NK). In order to confirm this hypothesis, we studied the production of cytotoxic effectors, such as perforin and granzymes, in CB NK cells. By flow cytometry analysis we observed that at 24h after treatment with IL-2, CB CD56+ cells (NK) showed a clear reduction of intracellular perforin and granzymes, as consequence of their release in the extracellular environment. To complete the analysis of cytotoxic profile of lymphocytes, we studied the production of an important mediator of cytotoxicity, the interferon-IFNIFNγ is a critical cytokine for innate and adaptive immunity against viral and bacterial infections and for tumor control. Flow cytometric analysis showed that expression of IFN was present in T lymphocytes and, moreover, different stimuli, such as IL-2, increased its expression. By contrast, NK cells were resulted IFNγ negative and unresponsive to IL-2 treatment (data contained in CD, Fig. A). lymphocytes and NK cells are two cell subsets involved mainly in innate immunity response against several pathogens, as bacteria and viruses. For this reason, we considered interesting to study the effect of viral infections, as HSV-1, on these two cell populations. Thus, aim of our project was to evaluate the activity of CB-derived T and NK lymphocytes in co-culture with HSV-1 infected cells. CB cells, that were previously stimulated with IL-2 and/or PAM for 14 days and then characterized phenotypically/quantitatively, were co-cultured with mock or HSV-1-infected human fibroblasts. At 24h after co-culture (48h after virus infection) we observed that the percentage of T lymphocytes did not change in the presence of uninfected and infected cells. Furthermore, T cells showed to have a non-effector phenotype (CD16-), independently co-culture conditions, suggesting that they did not exhibit a HSV-1 mediated response. Contextually, a significant reduction of non-cytotoxic phenotype of CB-derived V- T lymphocytes was observed after 24h of treatment with IL-2 or IL-2+PAM. These data confirmed our previous findings of increased cytotoxic activity of NK cells. Considering that CD16 is a marker of citotoxic activity of NK cells, we evaluated their response against HSV-1 infection, as cell expansion and cytokines production. Results showed that NK cell proliferation was induced by co-culture with human fibroblasts and greatly with HSV-1 infected human fibroblasts. By the contrast, IFN synthesized by NK cells was not modified by co-culture conditions, as well as the treatment with IL-2 or ABs. Differently, the co-culture with fibroblasts increased INFproduction by T lymphocytes. In order to verify if increased activation of CB immune system was able to contrast viral infection, we titrate the viral particles by cytopatic effect evaluation (TCID50). After 24h co-culture (48h after virus infection) we observed an increase in viral titer in co-culture with CB-derived cells IL-2 treated. This result could be explained as a consequence of viral particles released from fibroblasts lysed by cytolitic activity of NK cells. By the contrast, after 48h co-culture, the viral titer was reduced dramatically, as a result of lacked viral replication caused by cell death triggered by CB cells occured at 24h (preliminary data). Our conclusions have been suggested that T lymphocytes and NK cells cooperates to inhibit viral replication. T cells activated by IL-2 and/or ABs acquire a regulatory phenotype that allows them to produce immunoregulatory cytokines, such as INF, that consequently inhibits viral replication directly and promotes cytolytic activity of NK cells as confirmed by their ability to produce and release of perforin and granzymes, in response to a pathogen stimulus.