The IL-25/ILC2 axis promotes lung cancer with a concomitant accumulation of immune-suppressive cells in tumors in humans and mice.

Background: Group 2 innate lymphoid cells (ILC2) can be activated by interleukin (IL)-33 or IL-25. IL-25-activated ILC2 cells help protect the host against helminth infection while exacerbating allergic-like inflammation and tissue damage in the lung. In the context of cancer, IL-33-activated ILC2 cells were found to bear anti-tumoral functions in lung cancer while IL-25-activated ILC2 cells promoted tumorigenesis in colorectal cancer. The role of IL-25-activated ILC2 cells in lung cancer remains to be addressed. Methods: We examined the overall survival of human non-small cell lung cancer (NSCLC) patients according to IL25 expression as well as the distribution of ILC2 cells and regulatory T cells (Tregs) in various NSCLC patient tissues and peripheral blood (PB) of healthy donors (HDs). We analyzed the effect of adoptive transfer of IL-25-activated ILC2 cells on tumor growth, metastasis and survival in a heterotopic murine model of lung cancer. Results: We report that human NSCLC patients with high IL-25 expression have reduced overall survival. Moreover, NSCLC patients bear increased frequencies of ILC2s compared to HDs. Frequencies of Tregs were also increased in NSCLC patients, concomitantly with ILC2s. In mice bearing heterotopic lung cancer, adoptive transfer of IL-25-activated ILC2s led to increased tumor growth, increased metastasis and reduced survival. The frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs) were found to be increased in the tumors of mice that received ILC2s as compared to controls. Conclusion: Overall, our results indicate that the IL-25/ILC2 axis promotes lung cancer potentially by recruiting immune-suppressive cells to the tumors both in humans and in mice, and that it may therefore represent a suitable novel target for NSCLC immunotherapeutic development.

Cryopreservation of spleen and lymph nodes as a source of mononuclear cells to be used for the development of monoclonal antibody producing hybridoma cells.

In the present study, spleen and lymph nodes of mice were cryopreserved as a whole tissue and after thawing, membrane integrity of mononuclear cells was determined by trypan blue exclusion and PI staining. T and B lymphocytes, macrophages and dendritic cells have been isolated from both cryopreserved tissue and analyzed by Flow cytometry. BALB/c mice were immunized with Hepatitis e antigen (HBeAg) and spleen and lymph nodes of mice were cryopreserved for 3 to 10 months. The cells obtained from both tissue were applied to hybridoma technology to understand if the cells keep their viability and functionality. The cells were isolated and fused with F0 mouse myeloma cells and several antibody producing hybrid cells were developed. Results have shown that cryopreserved spleen and lymph nodes of mice can be efficiently used in hybridoma technology for the successful generation of monoclonal antibody producing hybrid cells.

Membrane-bound IL-15 stimulation on peripheral blood natural kiler progenitors leads to the generation of an adherent subset co-expressing dendritic cells and natural kiler functional markers.

Human peripheral blood natural killer progenitors represent a flexible, heterogeneous population whose phenotype and function are controlled by their membrane-bound IL-15. Indeed, reciprocal membrane-bond IL-15 trans-presentation commits these cells into NK differentiation, while membrane-bound IL-15 stimulation with its soluble ligand (sIL-15Rα) triggers a reverse signal (pERK1/2 and pFAK) that modifies the developmental program of at least two subsets of PB-NKPs. This treatment generates: i) the expansion of an immature NK subset growing in suspension; ii) the appearance of an unprecedented adherent non-proliferative subset with a dendritic morphology co-expressing marker, cytokines and functions typical of myeloid dendritic cells (CD1a(+)/BDCA1(+)/IL-12(+)) and NK cells (CD3-/NKp46(+)/ CD56(+)/IFNγ(+)). The generation of these putative NK/DCs is associated to the rapid inhibition of negative regulators of myelopoiesis (the transcription factors STAT6 and GATA-3) followed by the transient upregulation of inducers of myeloid development, such as the transcription factors (PU.1, GATA-1) and the anti-apoptotic molecule (MCL-1).

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

Differential effect of agonistic anti-CD40 on human mature and immature dendritic cells: the Janus face of anti-CD40.

Agonistic monoclonal antibodies to CD40 (CD40 mAbs) have a puzzling dual therapeutic effect in experimental animal models. CD40 mAbs induce tumor regression by potentiating antitumoral T-cell responses, yet they also have immunosuppressive activity in chronic autoimmune inflammatory processes. CD40 mAbs are thought to act on antigen presentation by dendritic cells (DCs) to T cells. DCs can be distinguished as either immature or mature by their phenotype and their ability to generate an effective T-cell response. Here we found that, on human cells, although anti-CD40 led immature DCs to mature and became immunogenic, it also reduced the capacity of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-matured DCs to generate a specific CD4 T-cell response. This inhibitory effect was related to rapid and selective apoptosis of mature DCs. Anti-CD40-mediated apoptosis was due to an indirect mechanism involving cooperation with the death domain-associated receptor Fas, leading to activation of Fas-associated death domain protein (FADD) and caspase-8. On human cells, CD40 activation by such agonists could, therefore, trigger immune responses to antigens presented by immature DCs, which are otherwise nonimmunogenic, by inducing maturation. On the other hand, anti-CD40 mAbs, by rapidly inducing apoptosis, may reduce the capacity of inflammatory signal-matured immunogenic DCs to generate an effective T-cell response. These results call for caution in CD40 mAb-based immunotherapy strategies.

Membrane-bound and soluble IL-15/IL-15Ralpha complexes display differential signaling and functions on human hematopoietic progenitors.

Membrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor alpha (Ralpha) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Ralpha complex with the IL-15Rbetagamma c chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1beta cells). This behavior is likely due to the IL-15Ralpha chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Ralpha complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rbetagamma c). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Ralpha/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Ralpha.

In human B cells, IL-12 triggers a cascade of molecular events similar to Th1 commitment.

Two functionally distinct subsets of B cells that produce Th1- and Th2-like patterns of cytokines have recently been identified. Interleukin-12 (IL-12) is a critical immunoregulatory cytokine that promotes Th1 differentiation through activation of signal transducer and activator of transcription 4 (STAT4). IL-12 has been reported to induce interferon gamma (IFN-gamma) production in B cells, but the relevant signaling pathways are poorly documented. Here, in human primary B cells, we found a functional IL-12 receptor (IL-12R) that internalizes following IL-12 binding. IFN-gamma and, to a lesser extent, IL-12 positively regulated the IL-12Rbeta2 subunit but had no effect on IL-12Rbeta1. On examining the effect of IL-12 on STAT4 and T-bet (2 key factors involved in IFN-gamma promoter activation), we found that IL-12 induced the phosphorylation and nuclear translocation of STAT4. IL-12-dependent constitutive STAT4 activation was also observed in the Epstein-Barr virus (EBV)-transformed B-cell line RPMI 8866 that spontaneously produces IL-12. T-bet expression has been shown to be dependent on STAT1. IL-12 had no direct effect on STAT1 activation or T-bet expression in primary B cells. In contrast, IL-12-induced IFN-gamma led to STAT1 activation, strong expression of T-bet, and IFN-gamma expression. IL-12 therefore initiates a cascade of events in B cells, including STAT4 activation, IL-12Rbeta2 up-regulation, IFN-gamma production, and T-bet up-regulation, potentially leading to Th1-like differentiation.

Critical role of JC virus-specific CD4 T-cell responses in preventing progressive multifocal leukoencephalopathy.

BACKGROUND: JC virus (JCV) is ubiquitous among the general population. However, only individuals with severely impaired immunity, mainly AIDS patients, develop progressive multifocal leukoencephalopathy (PML). Here, we examined the role of specific CD4 T cells in the control of JCV infection. METHODS AND DESIGN: JCV-specific CD4 T-cell responses were investigated by assaying peripheral blood mononuclear cell proliferation in response to the purified virus. Four groups of individuals without PML were examined: 14 HIV-seronegative healthy donors and 25 HIV-infected patients without PML, separated into urinary JCV excretors (active infection) and non-excretors, according to JCV PCR on urine. Two groups of patients with PML were also studied: 14 HIV-infected patients with active PML; and 10 PML survivors on effective and prolonged antiretroviral therapy. All of the patients were PCR-positive for JCV in the cerebrospinal fluid at the time of diagnosis of PML. RESULTS: No significant anti-JCV CD4 T-cell proliferation was found in any of the non-excretors tested. All nine healthy donors and seven of the 13 non-PML HIV-infected patients with urinary JCV excretion had positive JCV-specific CD4 T-cell responses. No significant response was found in the 14 patients with active PML, while nine of the 10 PML survivors had positive responses. Restoration of JCV-specific CD4 T-cell responses was associated with JCV clearance from the cerebrospinal fluid. CONCLUSION: JCV-specific CD4 T-cell responses appear to play a critical role in the control of JCV infection, preventing PML development. Such responses can be restored in PML survivors following effective and prolonged antiretroviral therapy.