It has been shown that γδ T cells protect against the formation of squamous cell carcinoma (SCC) in several models. However, the role of γδ T cells in human papillomavirus (HPV)-associated uterine cervical SCC, the third-leading cause of death by cancer in women, is unknown. Here, we investigated the impact of γδ T cells in a transgenic mouse model of carcinogenesis induced by HPV16 oncoproteins. Surprisingly, γδ T cells promoted the development of HPV16 oncoprotein-induced lesions. HPV16 oncoproteins induced a decrease in epidermal Skint1 expression and the associated antitumor Vγ5+ γδ T cells, which were replaced by γδ T-cell subsets (mainly Vγ6+ γδlowCCR2+CCR6-) actively producing IL-17A. Consistent with a proangiogenic role, γδ T cells promoted the formation of blood vessels in the dermis underlying the HPV-induced lesions. In human cervical biopsies, IL-17A+ γδ T cells could only be observed at the cancer stage (SCC), where HPV oncoproteins are highly expressed, supporting the clinical relevance of our observations in mice. Overall, our results suggest that HPV16 oncoproteins induce a reorganization of the local epithelial-associated γδ T-cell subpopulations, thereby promoting angiogenesis and cancer development.

Natural killer (NK) cells are cytotoxic lymphocytes and play a vital role in controlling viral infections and cancer. In contrast to B and T lymphopoiesis where cellular and regulatory pathways have been extensively characterized, the cellular stages of early human NK cell commitment remain poorly understood. Here we demonstrate that a Lin(-)CD34(+) CD38(+)CD123(-)CD45RA(+)CD7(+)CD10(+)CD127(-) population represents a NK lineage-restricted progenitor (NKP) in fetal development, umbilical cord blood, and adult tissues. The newly identified NKP has robust NK cell potential both in vitro and in vivo, generates functionally cytotoxic NK cells, and lacks the ability to produce T cells, B cells, myeloid cells, and innate lymphoid-like cells (ILCs). Our findings identify an early step to human NK cell commitment and provide new insights into the human hematopoietic hierarchy.

Virus‐like particles (VLPs) of human papillomavirus (HPV) are used as a vaccine against HPV‐induced cancer, and recently we have shown that these VLPs are able to activate natural killer (NK) cells. Since NK cells collaborate with dendritic cells (DCs) to induce an immune response against viral infections and tumors, we studied the impact of this crosstalk in the context of HPV vaccination. NK cells in the presence of HPV‐VLPs enhanced DC‐maturation as shown by an upregulation of CD86 and HLA‐DR and an increased production of IL‐12p70, but not of the immunosuppressive cytokine IL‐10. This activation was bidirectional. Indeed, in the presence of HPV‐VLPs, DCs further activated NK cells by inducing the upregulation of cell surface activation markers (CD69 and HLA‐DR). The function of NK cells was also improved as shown by an increase in IFN‐γ secretion and cytotoxic activity against an HPV+ cell line. This crosstalk between NK cells and DCs needed CD40 interaction and IL‐12p70 secretion, whereas NKG2D was not implicated. Our results provide insight into how VLPs interact with innate immune cells and how NK cells and DCs play a role in the immune response induced by this vaccine agent.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix, which mainly develops at the squamocolumnar (SC) junction. The progression of cervical HPV infections into (pre)neoplastic lesions suggests that viral antigens are not adequately recognized by innate immunity or presented to the adaptive immune system. Members of the defensin family have recently been found to inhibit viral and bacterial pathogens, to stimulate the migration of immune cells and to play a role in anticancer responses. In the present study, we focused on the poorly characterized human α-defensin 5 (HD-5) and its possible role in these processes. We showed that HD-5 was able to prevent HPV virion entry into cervical keratinocytes and to influence adaptive immunity. Indeed, this peptide specifically induced the chemoattraction and proliferation of both activated T lymphocytes and immature dendritic cells in a CCR2/CCR6-dependent manner and stimulated the infiltration of these professional antigen-presenting cells in a (pre)neoplastic epithelium transplanted in vivo in immunodeficient mice. No chemotactic effect was observed with plasmacytoid dendritic cells, macrophages or natural killer cells. Proliferative and angiogenic effects of HD-5 were also assessed in vitro and in vivo. However there was a striking regional disparity in expression of HD-5, being prominent in ectocervical, vaginal and vulvar neoplasia, while absent, or nearly so, in the cervical SC junction. Taken together, these results suggest one possible explanation for why the SC junction is uniquely vulnerable to both high-risk HPV infection (via reduced HD-5 expression and viral entry) and progression of neoplasia (via altered cell-mediated immune responses and altered microenvironment).

High-risk human papillomavirus infection is the etiological agent of cervical cancer, the third cause of cancer-associated death in women worldwide. Gamma delta T cells (γδ T cells) represent a small population of T cells expressing a T cell receptor (TCR) composed of gamma and delta chains. Their role in the context of HPV-induced lesions was not investigated yet, but we previously showed an infiltration of γδ T cells in this cancer, suggesting a relationship between HPV-induced lesions and γδ T cells. The goal of this project is to study the role of γδ T cells in the immune response against HPV-induced tumours. In order to study the role of γδ T cells in HPV-induced lesions, we have established a mouse model by crossing transgenic mice expressing HPV16 oncogenic genes, which develop spontaneous skin lesions, with γδ T cell-deficient mice. Surprisingly, depletion of γδ T cells significantly delays development of HPVinduced lesions. In parallel, we observed by immunohistochemistry an increase of leukocyte infiltration in HPV-induced lesions in absence of γδ T cells. Then, we evaluated by flow cytometry the proportions of immune cell populations present in the mouse skin and we found a larger proportion of CD4+ T cells in HPV and HPV γδ T cell-deficient mice compared to normal mice. Since γδ T cells could induce angiogenesis when infiltrating tumors, we measured blood vessels density in mice skin sections and we observed a significantly increase of blood vessels density in HPV mice compared to HPV γδ T cells-deficient mice. Our results suggest that γδ T cells could promote cancer progression in the context of HPV-induced lesions. We will further characterise these cells to understand in which cellular and molecular mechanisms they are involved.

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3-CD56+CD16+ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012). To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+/-CD127+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3-CD56+CD16+NKp46+ NK cells, CD19+ B, CD3+ CD4+ CD8+ T and CD33+CD14+ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γcnull (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKPs had a significant human cells engraftment and only CD3-CD56+NKp46+CD16+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34+ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate was also found in adult hBM and showed NK-lineage restriction. Our results indicate that the Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells. Disclosures: No relevant conflicts of interest to declare.

Historically, the name of natural killer (NK) cells came from their natural ability to kill tumor cells in vitro. From the 1970s to date, accumulating data highlighted the importance of NK cells in host immune response against cancer and in therapy-induced antitumor response. The recognition and the lysis of tumor cells by NK cells are regulated by a complex balance of inhibitory and activating signals. This review summarizes NK cell mechanisms to kill cancer cells, their role in host immune responses against tumor growth or metastasis, and their implications in antitumor immunotherapies via cytokines, antibodies, or in combination with other therapies. The regulatory role of NK cells in autoimmunity is also discussed.

Tracheobronchial squamous metaplasia is common in smokers, and is associated with both airway obstruction in chronic obstructive pulmonary disease (COPD) and increased risk of lung cancer. Although this reversible epithelial replacement is almost always observed in association with chronic inflammation, the role of inflammatory mediators in the pathogenesis of squamous metaplasia remains unclear. In the present study, we investigated the implication of cigarette smoke-mediated proinflammatory cytokine up-regulation in the development and treatment of tracheobronchial epithelial hyperplasia and squamous metaplasia. Using immunohistological techniques, we showed a higher epithelial expression of TNF-α, IL-1β, and IL-6, as well as an activation of NF-κB and activator protein-1/mitogen-activated protein kinase signaling pathways in the respiratory tract of smoking patients, compared with the normal ciliated epithelium of nonsmoking patients. In addition, we demonstrated that these signaling pathways strongly influence the proliferation and differentiation state of in vitro-generated normal human airway epithelial basal cells. Finally, we exposed mice to cigarette smoke for 16 weeks, and demonstrated that anti-TNF-α (etanercept), anti-IL-1β (anakinra), and/or anti-IL-6R (tocilizumab) therapies significantly reduced epithelial hyperplasia and the development of squamous metaplasia. These data highlight the importance of soluble inflammatory mediators in the pathogenesis of tracheobronchial squamous metaplasia. Therefore, the administration of proinflammatory cytokine antagonists may have clinical applications in the management of patients with COPD.

Human papillomavirus (HPV) infections account for more than 50% of infection‐linked cancers in women worldwide. The immune system controls, at least partially, viral infection and around 90% of HPV‐infected women clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and no study has evaluated the direct interaction between HPVs and NK cells, a key player in host resistance to viruses and tumors. We demonstrated an NK‐cell infiltration in HPV‐associated preneoplastic cervical lesions. Since HPVs cannot grow in vitro, virus‐like particles (VLPs) were used as a model for studying the NK‐cell response against the virus. Interestingly, NK cells displayed higher cytotoxic activity and cytokine production (TNF‐α and IFN‐γ) in the presence of HPV‐VLPs. Using flow cytometry and microscopy, we observed that NK‐cell stimulation was linked to rapid VLP entry into these cells by macropinocytosis. Using CD16+ and CD16− NK‐cell lines and a CD16‐blocking antibody, we demonstrated that CD16 is necessary for HPV–VLP internalization, as well as for degranulation and cytokine production. Thus, we show for the first time that NK cells interact with HPVs and can participate in the immune response against HPV‐induced lesions.