p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells.

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

Analysis of the thrombotic and fibrinolytic activities of tumor cell-derived extracellular vesicles.

Exosomes and microvesicles (MVs) are small extracellular vesicles secreted by tumor cells and are suggested to contribute to the thrombotic events that commonly occur in patients with advanced malignancies. Paradoxically, these vesicles have been reported to also possess fibrinolytic activity. To determine whether thrombotic or fibrinolytic activity is a predominant characteristic of these extracellular vesicles, we prepared exosomes and MVs from 2 breast cancer cell lines (MDA-MB-231 and MCF7), a lung cancer cell line (A549), and a leukemia cell line (NB4) and assayed their thrombotic and fibrinolytic activities. We observed that thrombotic activity was a common feature of MVs but not exosomes. Exosomes and/or MVs from several cell lines, with the exception of the A549 cell line, displayed fibrinolytic activity toward a pure fibrin clot, but only exosomes from MDA-MB-231 cells could degrade a fibrin clot formed in plasma. Increasing the malignant potential of MCF7 cells decreased the thrombotic activity of their MVs but did not alter their fibrinolytic activity. Decreasing the malignant potential of NB4 cells did not alter the thrombotic or fibrinolytic activity of their MVs or exosomes. Finally, the incubation of MDA-MB-231 cell-derived exosomes with A549 cells increased plasmin generation by these cells. Our data indicate that MVs generally have thrombotic activity, whereas thrombotic activity is not commonly observed for exosomes. Furthermore, exosomes and MVs generally do not display fibrinolytic activity under physiological conditions.

Fas Ligand Deficiency Impairs Tumor Immunity by Promoting an Accumulation of Monocytic Myeloid-Derived Suppressor Cells.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor-positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in naïve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumor-associated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells.

Implication of different effector mechanisms by cord blood-derived and peripheral blood-derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells ex vivo-expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56(+) CIK cells (CD3(+)CD56(+) and CD3(-)CD56(+)) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56(+) PB and CB-CIK cells to kill B-ALL cell lines have not been studied. METHODS: CB and PB-CIK cells were differentiated. CD56(+) CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines. RESULTS: We found that cytotoxic granule proteins were expressed at higher levels in CD56(+) PB-CIK than in CD56(+) CB-CIK cells. However, CD56(+) CB-CIK cells expressed more tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) compared with CD56(+) PB-CIK cells. We observed that CD56(+) CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56(+) PB-CIK cells that used only the NKG2D pathway. In contrast, CD56(+) PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56(+) CB-CIK cells used only the NKG2D pathway. CONCLUSIONS: Our results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.

Cord blood-derived and peripheral blood-derived cytokine-induced killer cells are sensitive to Fas-mediated apoptosis.

Fas-mediated apoptosis is one of the mechanisms used by tumor cells to escape the cytotoxicity of tumor-infiltrating lymphocytes. It has been suggested that cytokine-induced killer (CIK) cells are resistant to Fas-mediated apoptosis, thereby rendering them more attractive for use in cellular immunotherapy. Unlike what was observed by others, here we show that CIK cells are sensitive to Fas-mediated apoptosis. We have observed an increase in Fas expression in the different CIK cell subpopulations (CD3(+)CD56(-), CD3(+)CD56(+), and CD3(-)CD56(+)) isolated from both cord blood (CB) and peripheral blood (PB). We also show that the bulk, as well as the CD3(+)CD56(-) and CD56(+) CB- and PB-CIK cell subpopulations were sensitive to Fas-mediated apoptosis induced by both CH11 and APO-1 antibodies, albeit with a weaker effect for the CH11 antibody on CB-CIK cells. In addition, in the presence of the APO-1 and CH11 inducers, Fas engagement inhibited the cytotoxic activity of CB- and PB-CIK cells. This new contradictory result may help explain the variable efficacy observed with CIK cells in the clinic.

Human interferon-alpha increases the cytotoxic effect of CD56(+) cord blood-derived cytokine-induced killer cells on human B-acute lymphoblastic leukemia cell lines.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells may represent a promising immunotherapy for the treatment of children with relapsing B-lineage acute lymphoblastic leukemia (B-ALL) following hematopoietic stem cell transplantation (HSCT). Therefore, we investigated the possibility of combining adoptive immunotherapy with CIK cells and human interferon-alpha (hIFN-α) in order to potentiate the cytotoxicity of CIK cells against B-ALL. METHODS: Cord blood- derived CIK (CB-CIK) cells were differentiated, stimulated with phosphate-buffered saline (PBS) or hIFN-α, and tested for cytotoxic activity. We tested the anti-leukemic and graft-versus-host disease (GvHD) effects of CB-CIK cells in a human xenograft NOD/SCID/γc(-) (NSG) mouse model. RESULTS: Bulk CB-CIK cells showed very moderate cytotoxic activity while the subpopulation of CD56(+) CB-CIK cells showed significant cytotoxic activity against B-ALL cells. hIFN-α significantly augmented the cytotoxicity of CD56(+)CB-CIK cells in vitro and induced signal transducer and activator of transcription-1 (STAT1) phosphorylation. In addition, CD56(+)CB-CIK cells could delay mouse mortality significantly in vivo, and this effect was enhanced significantly by hIFN-α (P = 0.022). Furthermore, unlike CB mononuclear cells or peripheral blood mononuclear cells (PBMC), CD56(+)CB-CIK cells, alone or stimulated with hIFN-α, caused either no GvHD or mild GvHD, respectively, when injected into sublethally irradiated NSG mice. CONCLUSIONS: CD56(+)CB-CIK cells are effective cytotoxic agents against human B-ALL cell lines in vitro and possess anti-leukemic activity that is potentiated by hIFN-α in an NSG mouse model in vivo. These pre-clinical data support the testing of this immunotherapeutic approach in the clinic for the treatment of B-ALL.

Hepatitis C virus-specific cellular immune responses in individuals with no evidence of infection.

The detection of hepatitis C virus (HCV)-specific T cell responses in HCV-uninfected, presumably unexposed, subjects could be due to an underestimation of the frequency of spontaneously resolving infections, as most acute HCV infections are clinically silent. To address this hypothesis, HCV-specific cellular immune responses were characterized, in individuals negative for an HCV PCR assay and humoral response, with (n = 32) or without (n = 33) risk of exposure to HCV. Uninfected volunteers (n = 20) with a chronically HCV-infected partner were included as positive controls for potential exposure to HCV and HCV infection, respectively. HCV-specific T cell responses in freshly isolated peripheral blood mononuclear cells were studied ex vivo by ELISPOT and CFSE-based proliferation assays using panels of HCV Core and NS3-derived peptides. A pool of unrelated peptides was used as a negative control, and a peptide mix of human cytomegalovirus, Epstein-Bar virus and Influenza virus as a positive control. Overall, 20% of presumably HCV-uninfected subject tested had detectable T-cell responses to the virus, a rate much higher than previous estimates of HCV prevalence in developed countries. This result would be consistent with unapparent primary HCV infections that either cleared spontaneously or remained undetected by conventional serological assays.

Description of a new xenograft model of metastatic neuroblastoma using NOD/SCID/Il2rg null (NSG) mice.

In order to develop a relevant xenogenic animal model of neuroblastoma (NB), we compared the tumorigenicity and metastatic potential of SK-N-SH and SK-N-DZ NB cell lines in nude mice and NOD/SCID Il2rg null (NSG) mice. Subcutaneous injection of cell lines induced tumor formation only in NSG mice and was accompanied by metastasis to the liver, adrenal glands, skull and bone marrow. NSG mice injected intravenously showed a profile of distant metastasis that was not observed in nude mice. In addition, tumor growth rates and organ infiltration patterns associated with injected NB cell lines correlated with the in vitro proliferation properties and genetic markers of poor prognosis in NB patients. We also showed that cisplatin chemotherapy was able to inhibit tumor growth. These results clearly demonstrate the higher tumorigenic and metastatic potential of NB cells in NSG mice. Therefore, this xenograft NB model should prove useful in testing the efficacy of new therapeutic approaches for NB.