This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data ...show that CD56
CD16
and CD56
CD16
NK cells represent the predominant NK cell subpopulations in AML, while the CD56
CD16
NK cells are significantly reduced compared to HDs. Moreover, TIGIT
and PVRIG
cells cluster on the CD56
CD16
subset whereas CD39
and CD38
cells do so on CD56
CD16
NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.
Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3
T cells isolated from the ...peripheral blood (
= 20), malignant ascites (
= 16), and tumor tissue (
= 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8
T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1
CD8
T cell population was detected, which differed from PD-1
CD8
T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8
T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (
= 14) and malignant ascites (
= 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24
EpCAM
tumor cells showed a higher frequency of CD39
or CD73
cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8
T cells.
Leukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The ...present study provides an extensive characterization of macrophages in patients with acute myeloid leukemia (AML).
The phenotype and expression of coregulatory markers were assessed on bone marrow (BM)-derived LAM populations, using multiparametric flow cytometry. BM and blood aspirates were obtained from patients with newly diagnosed acute myeloid leukemia (pAML, n=59), patients in long-term remission (lrAML, n=8), patients with relapsed acute myeloid leukemia (rAML, n=7) and monocyte-derived macrophages of the blood from healthy donors (HD, n=17). LAM subpopulations were correlated with clinical parameters. Using a blocking anti-T-cell immunoreceptor with Ig and ITIM domains (TIGIT) antibody or mouse IgG2α isotype control, we investigated polarization, secretion of cytokines, and phagocytosis on LAMs and healthy monocyte-derived macrophages in vitro.
In pAML and rAML, M1 LAMs were reduced and the predominant macrophage population consisted of immunosuppressive M2 LAMs defined by expression of CD163, CD204, CD206, and CD86. M2 LAMs in active AML highly expressed inhibitory receptors such as TIGIT, T-cell immunoglobulin and mucin-domain containing-3 protein (TIM-3), and lymphocyte-activation gene 3 (LAG-3). High expression of CD163 was associated with a poor overall survival (OS). In addition, increased frequencies of TIGIT
M2 LAMs were associated with an intermediate or adverse risk according to the European Leukemia Network criteria and the FLT3 ITD mutation. In vitro blockade of TIGIT shifted the polarization of primary LAMs or peripheral blood-derived M2 macrophages toward the M1 phenotype and increased secretion of M1-associated cytokines and chemokines. Moreover, the blockade of TIGIT augmented the anti-CD47-mediated phagocytosis of AML cell lines and primary AML cells.
Our findings suggest that immunosuppressive TIGIT
M2 LAMs can be redirected into an efficient effector population that may be of direct clinical relevance in the near future.
Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in ...many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization.
We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium.
We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.
Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June ...2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913).
420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin FHA and pertactin PRN at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid TT and diphtheria toxoid DT), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination.
Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group).
This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.
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