B7-H6 as an efficient target for T cell-induced cytotoxicity in haematologic malignant cells.

T cells play crucial roles in the antitumour immune response. However, their dysfunction leads to inefficient tumour eradication. New members of the B7 family have moved to the fore of cancer research because of their involvement in T cell-mediated immune escape and tumorigenesis. Recently, bispecific antibodies (Bi-Abs) have become attractive because of their ability to activate T cells to target tumours. In this study, we examined the expression of new B7 family members B7-H4, B7-H5, B7-H6, and B7-H7 in human haematological tumour cells. Furthermore, we explored whether B7-H6 is an efficient target for T cell-induced cytotoxicity in haematologic malignant cells. We determined the capability of T cells armed with the bispecific antibody anti-CD3 × anti-B7-H6 (B7-H6Bi-Ab) to target haematological tumours in K562, Thp-1, Daudi, Jurkat, and U266 cells. Compared with their T cell counterparts, B7-H6Bi-Ab-armed T cells demonstrated significant cytotoxicity induction in B7-H6+ haematological tumour cells, according to quantitative luciferase and lactate dehydrogenase assays, and their activity was accompanied by increased levels of the secreted killing mediators granzyme B and perforin. Moreover, B7-H6Bi-Ab-armed T cells produced more T cell-derived cytokines: TNF-α, IFN-γ, and IL-2. In addition, compared to the control T cells, a higher level of the activation marker CD69 was detected on the B7-H6Bi-Ab-armed T cells. Taken together, these data suggest that the antitumour effect of B7-H6Bi-Ab-armed T cells may be a promising immunotherapy for use in future haematologic treatments.

Nanoparticle anchoring targets immune agonists to tumors enabling anti-cancer immunity without systemic toxicity.

Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)-2 generate effective anti-tumor immunity but also elicit serious toxicities, hampering their clinical application. Here we show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity, but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity, we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models, immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes, cytokine production, and granzyme expression, demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus, surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities.

Granzyme B producing B-cells in renal transplant patients.

OBJECTIVES: A separate subset of Granzyme B (GrB) producing B-cells regulating T-cell mediated immunity has been identified. In the present study, we investigated the role of GrB+ B-cells in renal transplant patients (RTX). METHODS: 12 healthy controls (HC) and 26 RTX patients were enrolled. In addition, 19 healthy volunteers treated with cyclosporine A (CsA) were enrolled. GrB+ B-cells were determined via flow cytometry. RESULTS: RTX Patients showed a diminished fraction of GrB+ B-cells as compared to HC. CsA treatment of healthy volunteers had no impact on the development of GrB+ B-cells. RTX patients with a history of allograft rejection showed an increased frequency of GrB+ B-cells. RTX patients with at least one episode of CMV viremia tended to have lower GrB+ B-cells as compared to patients without viremic episodes. CONCLUSION: We demonstrate that treatment with CsA does not impair the development of GrB+ B-cells. GrB+ B-cells may have a dual role in renal transplantation as regulatory cells to maintain allospecific tolerance and as effector cells enhancing viral control.

The effect of different anesthetics on tumor cytotoxicity by natural killer cells.

A number of retrospective studies have suggested that choice of anesthetic drugs during surgical tumor resection might affect tumor recurrence/metastasis, or outcome of patients. The recent study showed that volatile anesthetics-based general anesthesia was associated with the worse outcomes than intravenous anesthetics-based general anesthesia. However, the underlying mechanism is yet to be determined. Because natural killer (NK) cells are implicated as important immune cells for tumor recurrence/metastasis in the perioperative period, we examined the effect of different anesthetics on NK cell-mediated tumor cytotoxicity. Because adhesion molecule leukocyte function-associated antigen-1 (LFA-1) is functionally important in NK cells and is inhibited by commonly used volatile anesthetics isoflurane and sevoflurane, we hypothesized that these anesthetics would attenuate NK cell-mediated cytotoxicity. Using human NK cell line NK92-MI cells and tumor cell line K562 cells as a model system, we performed cytotoxicity, proliferation, conjugation and degranulation assays. Lytic granule polarization was also assessed. We showed that isoflurane, sevoflurane and LFA-1 inhibitor BIRT377 attenuated cytotoxicity, and reduced conjugation and polarization, but not degranulation of NK cells. Our data suggest that isoflurane and sevoflurane attenuated NK cell-mediated cytotoxicity at least partly by their LFA-1 inhibition in vitro. Whether or not isoflurane and sevoflurane attenuate NK cell-mediated tumor cytotoxicity in patients needs to be determined in the future.

Inhibition of Mammary Cancer Progression in Fetal Alcohol Exposed Rats by ß-Endorphin Neurons.

BACKGROUND: Fetal alcohol exposure (FAE) increases the susceptibility to carcinogen-induced mammary cancer progression in rodent models. FAE also decreases ß-endorphin (ß-EP) level and causes hyperstress response, which leads to inhibition of immune function against cancer. Previous studies have shown that injection of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) into the third ventricle increases the number of ß-EP neurons in the hypothalamus. In this study, we assessed the therapeutic potential of stress regulation using methods to increase hypothalamic levels of ß-EP, a neuropeptide that inhibits stress axis activity, in treatment of carcinogen-induced mammary cancer in fetal alcohol exposed rats. METHODS: Fetal alcohol exposed and control Sprague Dawley rats were given a dose of N-Nitroso-N-methylurea (MNU) at postnatal day 50 to induce mammary cancer growth. Upon detection of mammary tumors, the animals were either transplanted with ß-EP neurons or injected with dbcAMP-delivering nanospheres into the hypothalamus to increase ß-EP peptide production. Spleen cytokines were detected using reverse transcription polymerase chain reaction assays. Metastasis study was done by injecting mammary cancer cells MADB106 into jugular vein of ß-EP-activated or control fetal alcohol exposed animals. RESULTS: Both transplantation of ß-EP neurons and injection of dbcAMP-delivering nanospheres inhibited MNU-induced mammary cancer growth in control rats, and reversed the effect of FAE on the susceptibility to mammary cancer. Similar to the previously reported immune-enhancing and stress-suppressive effects of ß-EP transplantation, injection of dbcAMP-delivering nanospheres increased the levels of interferon-γ and granzyme B and decreased the levels of epinephrine and norepinephrine in fetal alcohol exposed rats. Mammary cancer cell metastasis study also showed that FAE increased incidence of lung tumor retention, while ß-EP transplantation inhibited lung tumor growth in both normal and fetal alcohol exposed rats. CONCLUSIONS: Our results suggest that increase of ß-EP production in the hypothalamus may serve as a potential therapeutic strategy for treating the cancer growth in patients with chronic stress and compromised immune function, such as the patients with FAE.

Fermented grape marc (FGM): immunomodulating properties and its potential exploitation in the treatment of neurodegenerative diseases.

The onset of neurodegenerative diseases has become more frequent than in the past also in relation to inappropriate dietary habits adopted in the western world. Nutraceuticals are currently investigated in order to prevent or retard the outcome of the so-called diet-related diseases, even including neurodegenerative pathologies. Here, we have in vitro studied the ability of fermented grape marc (FGM) from Negroamaro (N) and Koshu (K) Vitis vinifera to modulate the function of human peripheral blood mononuclear cells (PBMCs). Actually, both FGMs were able to increase the release and the intracellular content of inflammatory and anti-inflammatory cytokines, the induction of FoxP3 (a biomarker of T regulatory cells) and reduce the production of Granzyme B from PBMCs. Since these FGM-induced effects tend to polarize the immune response toward an anti-inflammatory pathway, the potential use of FGMs may represent a valid therapeutic measure to mitigating neuroinflammation in pathologies such as Parkinson disease and Alzheimer disease.

Increased natural killer T-like cells are a major source of pro-inflammatory cytokines and granzymes in lung transplant recipients.

BACKGROUND AND OBJECTIVE: Natural killer T (NKT)-like cells are a small but significant population of T lymphocytes; however, their role in lung transplant and the effect of current immunosuppressive agents on their function is largely unknown. We have previously shown lung transplant rejection was associated with an increase in peripheral blood T cell γ-interferon (IFN-γ), tumour necrosis factor-α (TNF-α) and granzyme B. NKT-like cells are a source of these pro-inflammatory mediators and as such may be involved in lung transplant pathology. METHODS: We analysed NKT-like cell numbers and cytokine and granzyme profiles in peripheral blood from a group of stable lung transplant patients and control subjects using multiparameter flow cytometry. RESULTS: There was a significant increase in NKT-like cells in transplant patients compared with control subjects (6.8 ± 4.9 vs 0.8 ± 0.2% lymphocytes respectively). There was an increase in the numbers of NKT-like cells producing IFN-γ, TNF-α, IL-2 IL-17, granzyme and perforin in transplant patients compared with controls. Immunosuppressant drugs were less effective at inhibiting IFN-γ and TNF-α production by T and NKT-like cells than NK cells in vitro. CONCLUSIONS: Current therapeutics is inadequate at suppressing NKT-like cell numbers and their production of pro-inflammatory mediators known to be associated with graft rejection. Alternative therapies that specifically target NKT-like cells may improve patient morbidity.

Cutting Edge: Responder T cells regulate human DR+ effector regulatory T cell activity via granzyme B.

MHC class II expression identifies an effector subset of human CD4(+)CD25(high)FoxP3(high) natural regulatory T cells (DR(+) Tregs) that induces more rapid suppression and exhibits higher FoxP3 expression than the remaining Treg population. Although Tregs are known to be highly sensitive to apoptosis, in this study we demonstrate that this sensitivity is primarily a feature of DR(+) Tregs. Granzyme B (GzmB) is strongly expressed by nonregulatory responder CD4 T cells, whereas effector DR(+) Tregs express little GzmB. Strong TCR stimulation markedly increases the expression of GzmB in all dividing responder CD4 T cells and mitigates the suppression by DR(+) Tregs. DR(+) Treg suppressive activity reemerges if GzmB is neutralized. We show that responder cells actively kill effector Tregs by producing GzmB in response to strong TCR stimulation. Thus, the production of GzmB by strongly activated CD4 T cells represents a mechanism by which CD4 T cells resist Treg suppression.

Etanercept reduces the serum levels of macrophage chemotactic protein-1 in patients with rheumatoid arthritis.

This study was performed to analyze the effect of etanercept, the soluble tumor necrosis factor-alpha (TNF-alpha) receptor, on the serum levels of several chemokines including monocyte chemotactic protein-1 (MCP-1), regulated upon activation normal T expressed and presumably secreted (RANTES), and granzyme B in rheumatoid arthritis (RA) patients. Twenty-eight patients with RA were administered etanercept once or twice a week for more than 6 months. Clinical and laboratory parameters were measured and serum levels of MCP-1, RANTES, and granzyme B were determined using enzyme-linked immunosorbent assay (ELISA) kits at baseline and at 3 and 6 months after the initial treatment. In addition, the levels of MCP-1, RANTES, and granzyme B produced by cultured synovial cells stimulated with TNF-alpha were measured. A significant decrease in serum MCP-1 levels was observed at 3 and 6 months after initial treatment with etanercept. Serum RANTES and granzyme B levels did not show significant changes. TNF-alpha induced MCP-1, RANTES, and granzyme B production in cultured synovial cells from RA patients. Serum MCP-1 levels were significantly correlated with the disease activity scores of 28 joints combined with CRP (DAS28-CRP), indicating the role of MCP-1 in the pathogenesis of rheumatoid inflammation. This study demonstrated that a reduction of MCP-1 production in RA patients was a newly determined effect of etanercept. Another cascade not associated with TNF-alpha may induce granzyme B and RANTES production in RA patients.

Heat shock protein 60 (HSP60) stimulates neutrophil effector functions.

Neutrophil granulocytes belong to the first cells that enter sites of infection, where they eliminate infiltrating pathogens via phagocytosis and the release of antimicrobial mediators. Hence, recruitment of neutrophils and activation of neutrophil microbicidal functions are crucial steps in the early containment of infection. In this study, we show that hHSP60 binds to murine and human PMN strongly and specifically. We demonstrate that HSP60 serves as a chemoattractant and modulates neutrophil functions. Human PMN were incubated with HSP60 alone or prior to stimulation with fMLP or PMA acetate. We observed that HSP60, although not inducing neutrophil release of ROS and degranulation itself, strongly enhanced the production of reactive oxygen induced by PMA and the release of primary granule enzymes induced by both secondary stimuli. This sensitization of PMN was HSP60-specific. Moreover, PMN that had been preincubated with HSP60 exhibited a marked increase in the uptake of opsonized Escherichia coli in the absence of additional stimuli. Taken together, our results show for the first time that HSP60 modulates antimicrobial effector functions of neutrophil granulocytes. In this way and in agreement with its function as an endogenous danger signal, HSP60, which is released by damaged tissue, may promote early innate defense mechanisms against invading pathogens.

An open-label, two-arm, phase I trial of recombinant human interleukin-21 in patients with metastatic melanoma.

PURPOSE: Human interleukin-21 (IL-21) is a pleiotropic class I cytokine that activates CD8(+) T cells and natural killer cells. We report a phase 1 study of recombinant human IL-21 in patients with surgically incurable metastatic melanoma. The primary objective was to investigate safety and tolerability by determining dose-limiting toxicity (DLT). The secondary objectives were to identify a dose response for various biomarkers in the peripheral blood, estimate the minimum biologically effective dose, determine the pharmacokinetics of IL-21, determine if anti-IL-21 antibodies were induced during therapy, and measure effects on tumor size according to Response Evaluation Criteria in Solid Tumors. EXPERIMENTAL DESIGN: Open-label, two-arm, dose escalation trial of IL-21 administered by i.v. bolus injection at dose levels from 1 to 100 microg/kg using two parallel treatment regimens: thrice weekly for 6 weeks (3/wk) or three cycles of daily dosing for 5 days followed by 9 days of rest (5+9). RESULTS: Twenty-nine patients entered the study. IL-21 was generally well tolerated and no DLTs were observed at the 1, 3, and 10 microg/kg dose levels. In the 3/wk regimen, DLTs were increased in alanine aminotransferase, neutropenia, and lightheadedness with fever and rigors. DLTs in the 5+9 regimen were increased in aspartate aminotransferase and alanine aminotransferase, neutropenia, fatigue, and thrombocytopenia. The maximum tolerated dose was declared to be 30 microg/kg for both regimens. Effects on biomarkers were observed at all dose levels, including increased levels of soluble CD25 and up-regulation of perforin and granzyme B mRNA in CD8(+) cells. One partial tumor response observed after treatment with IL-21 for 2 x 6 weeks (3/wk) became complete 3 months later. CONCLUSIONS: IL-21 is biologically active at all dose levels administered and is generally well tolerated, and phase 2 studies have commenced using 30 microg/kg in the 5+9 regimen.

Small-molecule Bcl-2 inhibitors sensitise tumour cells to immune-mediated destruction.

The cytotoxic effects of anticancer immune cells are mediated by perforin/granzyme-B, Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and therefore depend on intact apoptotic responses in target tumour cells. As killing by all three of these mechanisms is blocked by the frequently overexpressed antiapoptotic oncoprotein Bcl-2, we hypothesised that coexposure to a Bcl-2 inhibitor might enhance anticancer immune responses. We evaluated this in U937 lymphoma cells, and A02 melanoma cells, which both show strong Bcl-2 expression. Valpha24(+) Vbeta11(+) natural killer T (NKT) cells expanded from peripheral blood of normal donors (n=3) were coincubated with PKH26-labelled U937 cells, and cytotoxicity was determined by flow cytometry after annexin-V-FITC and 7-AAD staining. In all cases, addition of the HA14-1 small-molecule Bcl-2 inhibitor to the cocultures significantly increased apoptosis in the target U937 cells. Using a similar assay, killing of A02 cells by the cytotoxic T-lymphocyte clone 1H3 was shown to be amplified by coexposure to the potent small-molecule Bcl-2 inhibitor ABT-737. Experiments with immune effectors preincubated with concanamycin-A suggested that sensitisation to perforin/granzyme-B may underlie enhanced target-cell killing observed in the presence of Bcl-2 inhibitors. We conclude that immune destruction of malignant cells can be amplified by molecular interventions that overcome Bcl-2-mediated resistance to apoptosis.

Global transcriptional analysis delineates the differential inflammatory response interleukin-15 elicits from cultured human T cells.

OBJECTIVE: Interleukin 15 (IL)-15 controls proliferation and survival of T cells, but its effects and the underlying cellular regulation are not well understood. Previous studies have focused on its effects on short-term T-cell cultures. In view of the potential problems associated with using IL-2 alone in adoptive immunotherapy protocols, we investigated the impact of IL-15 on T-cell cultures and the global transcriptional effects it elicits in such cultures. MATERIALS AND METHODS: DNA microarrays and flow cytometry were used to examine the differential effect of 20 ng/mL IL-15 on primary serum-free T-cell cultures activated and cultured in the presence of IL-2. Quantitative reverse transcriptase polymerase chain reaction confirmed select microarray data. RESULTS: IL-15 significantly increased ex vivo expansion of primary human T cells over the entire 11-day expansions without affecting viability. The 1133 genes were consistently differentially expressed among three donor samples. Ontological analysis demonstrated that IL-15 increases expression of genes involved in inflammatory response (e.g., tumor necrosis factor [TNF]-alpha, Oncostatin M, CD40L, and CD33) and apoptosis (e.g., TNF-related apoptosis-inducing ligand). IL-15 also induced expression of four suppressors of cytokine signaling (SOCS) family genes (SOCS1-3, cytokine-inducible SH2-containing protein), which are classical negative regulators of cytokine signaling. IL-15 strongly suppressed the expression of inhibitory natural killer cell receptor genes, including three C-type lectins (KLRB1, KLRC1, and KLRD1), as well as IL-7Ra and Granzyme H. Finally, IL-15 induced differential expression of TNF receptor superfamily members (CD27 and CD30). CONCLUSION: These findings suggest that exogenous IL-15 may have a potential role in adoptive immunotherapy by both enhancing proliferation and modulating functionality during ex vivo T-cell expansion.

Lipophilic statins suppress cytotoxicity by freshly isolated natural killer cells through modulation of granule exocytosis.

NK cells, a component of the innate immune system, provide a first line of defense against viral infections and malignancies, interact with the adaptive immune system and have a role in rejection of allogeneic bone marrow transplants and solid allo- and xenotransplants. Immunoregulatory activity by the anti-hypercholesterolemia agents, 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) reductase inhibitors, known as statins, has recently been reported. We analyzed the effects of three statins on human NK cell cytotoxicity. Two lipophilic statins (simvastatin and fluvastatin) suppressed the cytotoxic activity of fresh and IL-2-stimulated NK cells, while pravastatin, a hydrophilic statin, did not. Suppression was not associated with changes in intracellular perforin, granzyme A or granzyme B levels, or with changes in expression of leukocyte function-associated antigen-1, an integrin known to regulate NK activity and reported to be altered by statin treatment. Decreased cytotoxicity was associated with decreased CD107a surface expression, indicating that the exocytosis pathway was compromised by simvastatin and fluvastatin but not by pravastatin. Mevalonate, the immediate downstream product of HMG-CoA reductase, partially reversed the effect of lipophilic statins on cytotoxicity and CD107a expression. Lipophilic statins also suppressed the release of the granule component, granzyme B, by IL-2-activated NK cells following stimulation with K562. That lipophilic statins suppress NK cell activity through inhibition of the exocytosis pathway suggest an additional potential role for statins in inhibition of transplantation responses.