Expansion and genetic modification of human natural killer cells for adoptive immunotherapy of cancer

A 
century 
after 
the 
initial
 proposition 
that 
the 
immune
 system 
has 
the
 capacity 
to
 fight
 against 
tumors, 
evading 
destruction
 by 
immune
 cells
 is 
now
 well 
recognized
 as
 a
 hallmark
 of
 cancer.
 Recent
 decades
 have
 witnessed
 extraordinary
 improvements 
in
 the 
use
 of
 immunotherapy 
against 
malignancies 
and
 adoptive
 transfer
 of
 Natural
 Killer
 (NK)
 cells
 stands
 among
 promising
 tools
 in
 the
 fight
 against
 cancer.
 Clinical
 studies
 have
 demonstrated
 the
 anti‐tumor
 responses
 generated 
by
 NK 
cells 
both 
in
 the
 autologous 
and
 allogeneic
 settings
 in
 various
 cancers.
Direct 
adoptive 
transfer,
 ex
 vivo
 activation 
and/o r
expansion, 
as 
well
 as
 genetic
 modification
 of
 NK
 cells
 aspire
 novel
 improvements
 to
 current
 immunotherapy
 strategies.
 As
 such
 interventions
 develop,
 the
 quest
 for
 better
 preparation
 of 
NK 
cell
 based 
therapies 
continues. This 
thesis, 
primarily
 investigates 
the
 feasibility
 and 
potential
 of
 ex
 vivo
 expanded
 NK
 cells
 for
 cancer 
immunotherapy. 
Our 
results
p roduced
 a 
system 
that
 has 
the
 capacity 
to
 expand
 polyclonal 
and 
highly 
cytotoxic
 NK 
cells showing 
selective
 anti‐ tumor
 activity.
 Protocols
 for
 expansion
 of
 these
 cells
 from
 healthy
 donors
 and
 patients
 with
 Multiple
 Myeloma
 (MM)
 using
 current
 Good
 Manufacturing
 Practice
 (cGMP)‐compliant
 methods
 have
 been
 optimized
 in
 conventional
 cell
 culture 
systems 
as
 well
 as
automated
 bioreactors.
The
 elevated
 cytotoxic
activity
 of 
expanded 
NK
 cells 
against
 autologous
 tumor 
cells,
 along
 with
 detailed
 analysis
 of
 phenotypic
 changes
 during
 the
 expansion
 process
 has
 subsequently
 shifted
 attention 
to
 the
 interaction 
between 
NK 
and
 tumor 
cells. Both 
as
 a 
basic 
method 
to
 identify
 these
 interactions,
 and 
as 
part 
of
 further
 plans
 to
 use
 genetically
 retargeted
 NK
 cells
 in
 cancer
 immunotherapy,
 we
 have
 investigated
 methods
 for 
efficient
 lentiviral
 genetic
 modification 
of
 NK 
cells.
This
 study 
has 
resulted 
in
 an
 optimized 
stimulation 
and
 genetic
 modification 
process
 for
 NK
 cells
 that
 greatly
 enhances
 viral
 gene
 delivery.
 Along
 with
 NK
 cell
 stimulating
 cytokines,
 an
 inhibitor
 of
 innate
 immune
 receptor
 signaling
 that
 blocks
 the
 intracellular
 detection
 of
 viral
 RNA
 introduced
 by
 the
 vector
 was
 successfully
 utilized 
to 
enhance 
gene
 transfer 
efficiency,
 also
 constituting 
a
 proof‐ of‐concept 
for
 various
 other
 gene
 therapy
 approaches. Taken
 together,
 the
 work
 presented
 in
 this
 thesis
 aims
 to
 bring
 us
 closer
 to
 optimal
 ex 
vivo
 manipulation 
of
 NK 
cells
 for
 immunotherapy.
 Clinical 
trials 
with
 the 
long‐term
 expanded 
NK 
cells
 as
 well
 as 
further 
preclinical 
development
 of 
NK
 cell
 genetic 
modification 
processes 
are
 warranted

Deletion of chromosomal region 8p21 confers resistance to Bortezomib and is associated with upregulated Decoy trail receptor expression in patients with multiple myeloma

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21)

Processing unit resources in a distributed computing systems

The described program is designed for collecting, storing and processing data of distributed file system

Plasmonic selection of ssDNA aptamers against fibroblast growth factor receptor

In this work, we describe the selection of ssDNA aptamers targeting fibroblast growth factor receptor binding protein 3 K650E, which has roles in cell division, growth, and differentiation through the kinase cascade. The selection process was based on the label-free, real-time monitoring of binding interactions by surface plasmon resonance, allowing for convenient manipulation of the selection rounds. Next generation sequencing data provided four major motif families from which nine individual sequences were selected based on their abundance levels. Electrophoretic mobility shift assays revealed binding of the selected aptamers to the target protein without significant interference from fibroblast growth factor receptor binding protein 2, indicating the selectivity of the aptamers. The dissociation constant at equilibrium for the best aptamer candidate, SU-3, was found to be (28.2 ± 19.6) × 10–9 M (n = 5) using a single-cycle kinetic analysis method. Advantages of the experimental setup and potential applications of the selected aptamers are discussed.Sabanci Universit

Correction: Characterization of zika virus infection of human fetal cardiac mesenchymal stromal cells.

[This corrects the article DOI: 10.1371/journal.pone.0239238.]

Myeloid maturation potentiates STAT3-mediated atypical IFN-γ signaling and upregulation of PD-1 ligands in AML and MDS.

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts' maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b fraction that expressed PD-1 ligands in response to IFN-γ. Intriguingly, STAT3 pathway was potently induced by IFN-γ and strengthened upon prolonged exposure. Nonetheless, STAT3-mediated atypical IFN-γ signaling appeared as a negligible factor for PD-L1 and PD-L2 expression. These negative influences of IFN-γ could be alleviated by a small-molecule inhibitor of STAT3, stattic, which also inhibited the upregulation of PD-L1. In conclusion, induction of myeloid maturation enhances the responsiveness of AML and MDS cells to IFN-γ. However, these malignant myeloid cells can exploit both STAT3 pathway and PD-1 ligands to survive IFN-γ-mediated immunity and maintain secondary immune resistance

Human immunodeficiency virus type 1 impairs sumoylation

During infection, the human immunodeficiency virus type 1 (HIV-1) manipulates host cell mechanisms to its advantage, thereby controlling its replication or latency, and evading immune responses. Sumoylation is an essential post-translational modification that controls vital cellular activities including proliferation, sternness, or anti-viral immunity. SUMO peptides oppose pathogen replication and mediate interferon-dependent anti-viral activities. In turn, several viruses and bacteria attack sumoylation to disarm host immune responses. Here, we show that HIV-1 impairs cellular sumoylation and targets the host SUMO E1-activating enzyme. HIV-1 expression in cultured HEK293 cells or in CD4(+) Jurkat T lymphocytes diminishes sumoylation by both SUMO paralogs, SUMO1 and SUMO2/3. HIV-1 causes a sharp and specific decline in UBA2 protein levels, a subunit of the heterodimeric SUMO E1 enzyme, which likely serves to reduce the efficiency of global protein sumoylation. Furthermore, HIV-1-infected individuals display a significant reduction in total leukocyte sumoylation that is uncoupled from HIV-induced cytopenia. Because sumoylation is vital for immune function, T-cell expansion and activity, loss of sumoylation during HIV disease may contribute to immune system deterioration in patients

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behcet's disease severity

Behcet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1 beta, IFN alpha, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis

Patients with 8p21 deletion respond poorly to bortezomib treatment.

<p>Patients diagnosed with MM and receiving bortezomib based treatment at 1^st line were selected to this study (n = 88). (<b>A)</b> left panel is the non-HDT patients that received bortezomib (Vel: Velcade®, n = 33) as 1^st line treatment and right panel is the non-HDT patients that did not respond to 1^st line bortezomib treatment or relapsed and treated with Lenalidomide based regimen as a 2^nd line treatment (n = 16). B. Left panel displays patients received high dose treatment and bortezomib as 1^st line treatment (n = 55) and right panel is the response to Lenalidomide based treatment as 2^nd line for relapse patients (n = 18). Details of the treatments are shown in materials & methods section. Chi-square test was performed to analyze results.</p