Anti-cancer γδ T lymphocytes: contradictory past and promising future

Recent anti-cancer strategies are based on the stimulation of anti-tumor immune reaction, exploiting distinct lymphocyte subsets. Among them, γδ T cells represent optimal anti-cancer candidates, especially in those tissues where they are highly localized, such as the respiratory or gastrointestinal tract. One important challenge has been the identification of stimulating drugs able to induce and maintain γδ T cell-mediated anti-cancer immune response. Amino-bisphosphonates (N-BPs) have been largely employed in anti-cancer clinical trials due to their ability to upregulate the accumulation of pyrophosphates that promote the activation of Vγ9Vδ2 T cells. This activation depends on the butyrophilin A family, which is crucial in contributing to Vγ9Vδ2 T cells stimulation but is not equally expressed in all cancer tissues. Thus, the clinical outcome of such treatments is still a challenge. In this viewpoint, a critical picture of γδ T cells as effective anti-cancer effectors is designed, with a specific focus on the best immune-stimulating therapeutic schemes involving this lymphocyte subset and the tools available to measure their efficacy and presence in tumor tissues. Some pre-clinical models, useful to measure γδ T cell anti-cancer potential and their response to stimulating drugs, therapeutic monoclonal antibodies, or bispecific antibodies are described. Computerized imaging and digital pathology are also proposed as a help in the identification of co-stimulatory molecules and localization of γδ T cell effectors. Finally, two types of novel drug preparation are proposed: nanoparticles loaded with N-BPs and pro-drug formulations that enhance the effectiveness of γδ T lymphocyte stimulation

HIV-1 Tat Triggers TGF-β Production and NK Cell Apoptosis that is Prevented by Pertussis Toxin B

Herein, we show that PTX-B and its non-toxic mutant PT9K/129G inhibit transcription and secretion of TGF-β elicited by HIV-1 Tat in NK cells. Moreover, Tat strongly activates the cJun component of the multimolecular complex AP-1, while TGF-β triggers cFos and cJun. Treatment of NK cells In turn,with PTX-B or PT9K/129G inhibits Tat and TGF-β-induced activation of AP-1. TGF-β enhances starvation-induced NK cell apoptosis, reduces the transcription of the antiapoptotic protein Bcl-2 and inhibits Akt phosphorylation induced by oligomerization of the triggering NK cell receptor NKG2D. All these TGF-β-mediated effects are prevented by PTX-B or PT9K/129G, through a PI-3K-dependent mechanism. Finally, PTX-B and PT9K/129G upregulate Bcl-xL, the isoform of Bcl-x that protects cells from starvation-induced apoptosis. Of note, in NK cells from patients with HIV-1 infection, mRNA expression of Bcl-2 and Bcl-xL was consistently lower than that of healthy donors; interestingly, TGF-β and Tat were detected in the sera of these patients. These data suggest that Tat-induced TGF-β production and the consequent NK cell failure, possibly occurring during early HIV-1 infection, may be regulated by PTX-B and PT9K/129G

Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR) and NKG2D

Human natural killer (NK) lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS) members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis). Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR) NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity

Stress immunity in lymphomas: mesenchymal cells as a target of therapy.

Abstract The role of lymphocytes in eliminating lymphoma cells is based on the interaction between activating receptors on lymphocytes and target surface ligands on lymphoma cells. Stress-related immunity can be triggered both in Hodgkin's (HL) and non-Hodgkin lymphomas (NHL), through the activation of the NKG2D receptor on CD8+ T and gammadelta T lymphocytes, by NKG2D-ligands (NKG2D-L), as the MHC class-I related molecules MIC-A/B and the UL16-binding proteins 1-4 (ULBPs), expressed on lymphoma cells. Furthermore, NKG2D-L can be shed and interact with NKG2D on effector lymphocytes affecting the recognition of lymphoma cells. Proteolytic cleavage of MIC-A is known to depend on the thiol isomerase ERp5 and the disintegrins and metalloproteinases ADAM10 and ADAM17, which also cleave ULPBs. Mesenchymal stromal cells (MSC) are relevant in regulating effector T lymphocytes-mediated lymphoma surveillance. Indeed, MSC can be seen as targets of potential new therapeutic schemes acting on lymphoma microenvironment, to redirect the stress immune response and avoid escape strategies, by inducing stress molecules, inhibiting sheddase activity, shifting cytokine production to Th1 pattern and blocking Treg differentiation

Imatinib mesylate can help to direct natural immunity toward an anti-leukemic reactivity by acting on the bone marrow microenvironment

We discuss our recent findings on the increase, in chronic myeloid leukemia patients treated with imatinib, of B1 lymphocytes producing IgM anti-O-linked sugars expressed by leukemic cells, paralleled by increased B-stimulating cytokines. We propose that one important effect of imatinib treatment is due to the remodelling of bone marrow microenvironment

Generation of CD4+ or CD8+ regulatory T cells upon mesenchymal stem cell-lymphocyte interaction

Background and Objectives Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based on their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in co-cultures of peripheral blood mononuclear cells (PBMC) and MSC.Design and Methods MSC were obtained from the bone marrow of four healthy donors and nine patients with acute leukemia in complete remission following chemotherapy. Short-term (4 days) co-cultures of MSC and autologous or allogeneic PBMC were set up, the lymphocytes harvested and their regulatory activity assessed.Results Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or phytohemagglutinin-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Regc), were all CD45+CD2+ with variable proportions of CD25+ cells (range 40–75% n=10) and a minor fraction expressed CTLA4 (2–4%, n=10) or glucocorticoid-induced tumor necrosis factcor receptor-related gene (0.5–4% n=10). Both CD4+ and CD8+ Regc purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Regc:responder cell ratio. CD4+ Regc expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8+ Regc did not. The effectiveness of Regc, whether CD4+ or CD8+, was 100-fold higher than that of CD4+CD25+high regulatory T cells. Regc were also generated from highly purified CD25− PBMC or CD4+ or CD8+ T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-β and prostaglandin E2 did not appear to be involved in the generation of Regc or in the Regc-mediated immuno-suppressive effect. Furthermore, cyclosporine A did not affect Regc generation or the immunosuppression induced by Regc.Interpretation and Conclusions These findings indicate that powerful regulatory CD4+ or CD8+ lymphocytes are generated in co-cultures of PBMC with MSC. This strongly suggests that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect

Interaction between Human NK Cells and Bone Marrow Stromal Cells Induces NK Cell Triggering: Role of NKp30 and NKG2D Receptors

Abstract In this study we have analyzed the interaction between in vitro cultured bone marrow stromal cells (BMSC) and NK cells. Ex vivo-isolated NK cells neoexpressed the activation Ag CD69 and released IFN-γ and TNF-α upon binding with BMSC. Production of these proinflammatory cytokines was dependent on ligation of ICAM1 expressed on BMSC and its receptor LFA1 on NK cells. Furthermore, the NKp30, among natural cytotoxicity receptors, appeared to be primarily involved in triggering NK cells upon interaction with BMSC. Unexpectedly, autologous IL-2-activated NK cells killed BMSC. Again, LFA1/ICAM1 interaction plays a key role in NK/BMSC interaction; this interaction is followed by a strong intracellular calcium increase in NK cells. More importantly, NKG2D/MHC-I-related stress-inducible molecule A and/or NKG2D/UL-16 binding protein 3 engagement is responsible for the delivery of a lethal hit. It appears that HLA-I molecules do not protect BMSC from NK cell-mediated injury. Thus, NK cells, activated upon binding with BMSC, may regulate BMSC survival

Discovery of a new selective inhibitor of A Disintegrin And Metalloprotease 10 (ADAM-10) able to reduce the shedding of NKG2D ligands in Hodgkin's lymphoma cell models

Hodgkin's lymphoma (HL) is the most common malignant lymphoma in young adults in the western world. This disease is characterized by an overexpression of ADAM-10 with increased release of NKG2D ligands, involved in an impaired immune response against tumor cells. We designed and synthesized two new ADAM-10 selective inhibitors, 2 and 3 based on previously published ADAM-17 selective inhibitor 1. The most promising compound was the thiazolidine derivative 3, with nanomolar activity for ADAM-10, high selectivity over ADAM-17 and MMPs and good efficacy in reducing the shedding of NKG2D ligands (MIC-B and ULBP3) in three different HL cell lines at non-toxic doses. Molecular modeling studies were used to drive the design and X-ray crystallography studies were carried out to explain the selectivity of 3 for ADAM-10 over MMPs