Engagement of NKG2D by their ligands (NKG2D-L), as the human major histocompatibility complex class I-related molecules MIC-A and the UL16-binding proteins, on cytolytic lymphocytes leads to the ...enhancement of antitumour effector functions. These ligands are missing or expressed at very low levels on leukaemic cells; furthermore, they can be shed by tumour cells and inhibit cytolytic activity of lymphocytes. Herein, we show that in vivo administration of all-trans-retinoic acid (ATRA) or the histone deacetylase inhibitor sodium valproate (VPA) to patients affected with acute myeloid leukaemia (AML) M3 or M1 respectively, leads to the induction of transcription and expression of NKG2D-L at the surface of leukaemic cells. Apparently, no detectable shedding of the soluble form of these molecules was found in patients' sera. Conversely, AML blasts from patients treated with chemotherapy not including ATRA or VPA did not show any induction of NKG2D-L transcription. Furthermore, upon therapy with ATRA or VPA, leukaemic blasts become able to trigger lytic granule exocytosis by autologous CD8(+) T and natural killer lymphocytes, as shown by CD107a mobilization assay, followed by leukaemic cell lysis. These findings indicate that ATRA and VPA may contribute to the activation of cytolytic effector lymphocytes in vivo, possibly enhancing their anti-leukaemic effect.
...it is also relevant to point out that the infiltration of neoplasia as breast and gastrointestinal tract cancer with lymphocytes could identify patients with a better outcome 9,10 . ...a therapy ...which favours the anti-tumour immune response could be an additional weapon against cancer. Many inhibiting factors responsible for the MSC-mediated downregulation of effect or lymphocyte activation ( Figure 1) have been identified including TGFβ, IL10 and prostaglandin E2, indoleamine 2,3 dioxigenase, heme oxigenase, arginase 1 and 2, nitric oxidase synthase 2, hepatocyte growth factor and peculiar histocompatibility antigens such as HLAG5 13,14 . ...it is conceivable that targeting of CAF-derived immunosuppressive factors can be a tool to favour anti-tumour immune response. Conclusions MSCs localized in tumour microenvironment can promote through TGFβ the EMT that facilitate the generation of metastasis and can downregulate innate and adaptive immune responses. ...MSC can be a suitable target for tumour therapy both using drugs which impair the MSC-mediated inhibitions by eliminating MSC or try to switch the MSC-mediated immune suppression into an activation.
Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based of their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in ...co-cultures of peripheral blood mononuclear cells (PBMC) and MSC.
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.
Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or PHA-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-b and prostaglandin E2 did not appear to be involved in the generation of Regc or in the Regc-mediated immunosuppressive effect. Furthermore, cyclosporin A did not affect Regc generation or the immunosuppression induced by Regc.
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.
There is increasing evidence that statins, inhibitors of 3-hydroxyl-3-methylglutaryl coenzyme A reductase, can effectively be used not only in the treatment of hypercholesterolemia, but also in other ...human disorders; indeed, statins have strong anti-inflammatory and immunomodulatory effects, so that they can influence the onset and outcome of inflammation and autoimmunity. On the other hand, it has been shown that statins can affect growth and survival of solid tumour and leukemic cells, thus they have been proposed in the treatment of neoplasias as multiple myeloma, in association with drugs, as thalidomide, known to act on the cancer microenvironment. In the current view, tumor microenvironment include many cell types that interact with tumor cells: among them, stromal and endothelial cells, macrophages and dendritic cells, the various types of lymphocytes such as NK cells, B and T cells. The interplay between all these cell populations, and the balance between these, determines whether there is a tumour cell growth promotion or inhibition. In haematological malignancies, such as multiple myeloma, chronic lymphocytic and myeloid leukemias and follicular lymphomas, the survival, drug-resistance and proliferation of leukemic cells have been shown to be largely dependent on a supportive microenvironment, so that some cellular components of it, mainly mesenchymal stromal cells, cancer associated fibroblasts and macrophages, are now proposed as targets of new therapies. Herein, we analyze the effects that statins can exert on cancer cells, stromal cells and human natural killer cells, to discuss whether they can be proposed as anti-cancer drugs.
In this study, we analysed 30 patients with B-cell chronic lymphocytic leukaemia (CLL), compared with 10 healthy donors, for the expression and function of the leukocyte-associated Ig-like receptor-1 ...(LAIR-1). LAIR-1 is an inhibitory receptor containing a cytoplasmic tyrosine-based inhibitory motif (ITIM) that binds to the SH2 domain of phosphatases, leading to dephosphorylation of different kinases. Constitutive activation of c-Jun aminoterminal kinase (JNK), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase, has been reported in CLL. We show that LAIR-1 is absent in high-risk (HR) CLL and differently expressed on intermediate- and low-risk CLL and the intensity of expression, which is always significantly lower than in healthy donors, correlates with disease stage and progression. Interestingly, both constitutive and sIgM-induced phosphorylation of p38 and JNK is inhibited by LAIR-1 through an ITIM-dependent signal, as demonstrated by the use of specific ITIM-binding peptides; importantly, this inhibitory signal is missing when LAIR-1 is not expressed as occurs in HR CLL. Moreover, engagement of LAIR-1 blocks constitutive and sIgM-induced Akt phosphorylation, besides nuclear factor kappa-B nuclear translocation, and prevents CLL division. These results suggest that CLL lacking LAIR-1 may miss one of the molecular mechanisms controlling B-cell activation and proliferation.
ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been ...reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naïve, germinal center and memory B cells from tonsils, CD19+ CD5+ cells from cord blood and CD19+ lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.
Down-regulation of expression of MHC alleles, as well as tumor-specific antigens, is observed frequently during tumor progression, resulting in an impairment of MHC-restricted, alpha beta ...-T-cell-mediated, tumor-specific immunity. Given the unique set of antigens recognized and the lack of requirement for classical antigen-presenting molecules, gamma delta T cells might, therefore, represent a nonredundant system in anticancer surveillance, as proposed for the immune response against pathogens. Evidence that gamma delta and alpha beta T cells make distinct contributions to anticancer surveillance has been provided recently in mice. Here, we discuss the potential role played by resident V delta 1 super(+) and circulating V delta 2 super(+) T cells in the defense against solid tumors and hematological malignancies.
In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin ...and granzymes, and IFN-gamma secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-gamma release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.
It has been suggested that members of HSP families represent the surface target of immune responses leading to tumor rejection in mice. Here we report that tumor cells, compared with normal cells, ...constitutively expressed 2- to 10-fold higher levels of intracellular HSP90. Moreover, in the absence of environmental stress, 2 lines (out of 6) expressed the "inducible" HSP72, which was also detectable in fresh tumor cells. HSP72 expression was not regulated during the cell cycle, in contrast with what has been observed with normal cells. Both HSP90 and HSP72 proteins exhibited a heterogeneous pattern of intracellular distribution in most cells, HSP72 being confined mainly to the nuclear compartment. Finally, we could detect both HSP90 and, to a lesser extent, HSP72 (that are generally believed to be located intracellularly) at the surface of some tumor cell lines. We conclude that tumor cells differ from normal cells in their pattern of HSP expression; this might imply a role of HSPs in eliciting an immune response against cancer.
Bone marrow mesenchymal stromal cells can suppress T-lymphocyte proliferation but promote survival of normal and malignant B cells, thus representing a possible target for new therapeutic schemes. ...Here we defined the effects of cholesterol synthesis inhibitors on the interaction between these mesenchymal stromal cells and T or B lymphocytes.
We exposed mesenchymal stromal cells to inhibitors, such as fluvastatin, of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, responsible for the synthesis of mevalonate, the precursor of cholesterol. Also, these cells were treated with manumycin A, a farnesyl transferase inhibitor which blocks the mevalonate-dependent isoprenylation of small guanosin triphosphate binding proteins. First, mesenchymal stromal cell morphology, cytoskeleton assembly, cell cycle, survival and cytokine production were evaluated. Then, these cells were co-cultured with either T or B lymphocytes and we analyzed: 1) the inhibition of T-cell proliferation to mitogenic stimuli; 2) B-cell survival.
Fluvastatin altered the assembly of actin microfilaments, inactivated RhoA guanosin triphosphate binding protein, inhibited the S-phase of the cell cycle, induced apoptosis in a small fraction of cells but preserved cytokine production. Preincubation of mesenchymal stromal cells with fluvastatin, or manumycin A, down-regulated the expression of adhesion molecules, reduced cell-to-cell interactions and prevented the inhibition exerted by these stromal cells on CD3/T-cell receptor-induced lymphocyte proliferation. Mevalonic acid could revert morphological, phenotypic and functional effects of fluvastatin. Finally, fluvastatin significantly reduced the mesenchymal stromal cells-mediated rescue of B cells in the presence of dexamethasone, although it did not function in the absence of corticosteroids.
Fluvastatin-mediated effects on bone marrow mesenchymal stromal cells were conceivably due to the inhibition of isoprenylation of small guanosin triphosphate binding proteins, occurring for the lack of mevalonate. Altogether these findings suggest that drugs acting on the mevalonate biosynthetic pathway can regulate mesenchymal stromal cell-induced T-cell suppression and B-lymphocyte survival.
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