Download Immune system fighting malignancy

Vol 17 – 1/2016
Immune system
fighting malignancy
Antigen-specific naive
and memory T cells
Direct phenotypic and
functional characterization
p. 11
Evaluation of
metastatic burden
Recovery of human metastatic
cells from mouse model
p. 17
DC vaccination
Designing a DC-based therapy
for primary liver cancer
p. 20
CONTENTS
Editorial
3
News
Commercial-scale manufacture of genetically modified T cells – challenges and approaches
4
Reports
Dendritic cells pulsed with PepTivator® Ovalbumin induce both OVA-specific CD4+ and CD8+ T cells and cause antitumor
effects in a mouse model of lymphoma
7
Kenji Miki, Koji Nagaoka, Hermann Bohnenkamp, Takayuki Yoshimoto,
Ryuji Maekawa, and Takashi Kamigaki
Detection, enrichment, and direct phenotypic and functional characterization of antigen-specific naive
and memory T cell subsets
11
Petra Bacher and Alexander Scheffold
Evaluation of metastatic burden and recovery of human metastatic cells from a mouse model
17
Lorena Landuzzi, Arianna Palladini, Marianna Lucia Ianzano, Roberta Laranga,
Giulia D’Intino, Patrizia Nanni, and Pierluigi Lollini
Perspectives
Designing a dendritic cell–based therapy for primary liver cancer
20
Stuart M. Curbishley, Miroslava Blahova, and David H. Adams
Engineering human cells with lentiviral vectors: Making an impact on human disease
25
Rimas Orentas
2
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Vol 17 • 1/2016
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EDITORIAL
Dear Researcher,
The immune system with its vast array of different cell types has a myriad of ways to protect the
body against malignant disease. Researchers and clinicians worldwide explore the potential of
these cells in their quest for novel cellular therapies. In this MACS&more issue, scientists from
around the world share their research results and experience on the way to translating basic
research into clinical application.
With the development of new strategies for
cellular therapy, the scientific community has
gained enormous momentum in the fight
against malignancy. Particularly, genetically
engineered T cells expressing a chimeric
antigen receptor (CAR) show great promise
and – in the future – might provide the basis
for the treatment of diseases that are currently
considered incurable. On p. 25 Rimas Orentas
provides an exciting perspective on lentiviral
technology, which is one of the cornerstones
of successful CAR T cell manufacture, and on
future directions for CAR T cell engineering.
Another very promising immunotherapy
approach is the vaccination with tumor
antigen–presenting dendritic cells (DCs),
which can initiate an effective anti-cancer
immune response in the body. Using a mouse
model of lymphoma, Kenji Miki et al. showed
that DCs pulsed with a PepTivator® Peptide
Pool induced both CD4+ and CD8+ T cells to
proliferate and release cytokines. Ultimately,
the DCs inhibited tumor growth (p. 7).
Stuart Curbishley gives thorough insight into
the process of designing a randomized phase
II clinical trial utilizing DCs as vaccines for
hepatocellular carcinoma (p. 20). Aim of the
study is to evaluate whether vaccination with
DCs provides an additional benefit compared
to cyclophosphamide pre-conditioning
and transarterial chemoembolization alone.
Throughout the entire DC manufacturing
workflow, S. Curbishley and his colleagues
rely on a wide range of products from
Miltenyi Biotec.
miltenyibiotec.com
Petra Bacher and Alexander Scheffold
(p. 11) established a technique based on MACS®
Technology to increase the sensitivity for flow
cytometry analysis of rare antigen-specific
T cells. The authors also set up a multicolor
panel using numerous MACS Antibodies.
The so-called ARTE (antigen-reactive T cell
enrichment) together with multicolor flow
cytometry will be an excellent tool for research
into various immune-related diseases and the
development of immunotherapies.
The spread of malignant cells in the body
is an important topic of cancer research as
metastases are the major cause of death in
cancer-related diseases. Lorena Landuzzi et al.
describe a method that allows the recovery and
quantification of human metastatic cells in a
mouse model and enables enrichment of these
cells for in vitro analyses (p. 17).
These are just a few examples of how Miltenyi
Biotec’s comprehensive, integrated portfolio
has supported researchers on the journey
towards novel cellular therapies against
malignant disease.
We hope you enjoy reading this MACS&more
issue!
Your MACS&more team
Vol 17 • 1/2016
MACS & more
3
NEWS
Commercial-scale manufacture of genetically
modified T cells – challenges and approaches
Adoptive transfer of genetically modified T cells holds great promise for cancer therapy. The manufacture of these cells however is complex,
labor-intensive, and comprises many different handling steps – a challenge for the conversion from small-scale clinical trials into larger
commercial-scale treatments.
The CAR T cell manufacturing
process – complex and challenging
T cells play a pivotal role in the immune
response against cancer. Accordingly, the
capacity of T cells to fight malignant diseases
provides exciting perspectives with regard to
novel, widely applicable cell therapy options.
In fact, the adoptive transfer of T cells
expressing genetically engineered chimeric
antigen receptors (CARs)¹ has shown great
promise in clinical studies addressing chronic
or acute lymphocytic leukemia²,³. However,
the preparation of CAR T cells for clinical
application is quite complex and comprises
numerous handling steps, including i)
enrichment of the T cells, ii) T cell activation,
iii) transduction, iv) expansion, and finally
v) cell formulation (fig. 1). In the context of
a small-scale clinical trial, all these steps can
be performed reasonably according to GMP
guidelines in a semi-automated manner using
several devices and a multitude of liquid
handling steps. However, the transformation
of such manufacturing methods into a largescale setting has some pitfalls. The complex
procedure includes a number of processes that
are usually performed in an open environment,
thus entailing high demands on the clean
room infrastructure and on skill and time for
the personnel. Currently, only few institutions
would be able to fulfill these requirements,
which limits the number of cellular products
that can be manufactured or prepared within
a specified time. Ultimately, this also narrows
the number of patients who could benefit from
such therapies. To make cell–based therapy
available for many patients, cell manufacture
processes need to be adapted and optimized to
a commercial scale.
4
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Vol 17 • 1/2016
Donor/
patient
Cryo-preservation
Blood
leukapheresis
T cell
selection
Activation
Transduction
Enrichment
of modified
T cells
Cryo-preservation
Expansion
Final
formulation
Administration
to patient
Figure 1 Workflow for the production of genetically engineered T cells.
There are quite a few needs that a process for
manufacturing a cellular therapy product has
to fulfill. First and foremost, the resulting
cellular product must be safe and clinically
effective (meaning the cells must meet certain
functional requirements). Moreover, the
GMP-compliant manufacturing process has
to be reliable and robust to yield a product
with consistent quality, which can be easily
validated. For commercial-scale manufacture,
it is also necessary to optimize the process with
regard to labor intensity, simplification of the
workflow, and scalability of production.
clean room requirements. Importantly, all cell
processing steps are automated, which not only
makes for a highly reproducible, standardized,
and robust manufacturing process, it also
reduces labor intensity.
The CliniMACS Prodigy® – enabling
robust end-to-end GMP-compliant
manufacture of CAR T cells
Miltenyi Biotec has developed the CliniMACS
Prodigy® as an all-in-one solution for cell
processing in a closed GMP-compliant system.⁴
An automated process specifically developed
and optimized for the manufacture of CAR
T cells will be available soon. With this process,
the entire workflow for the manufacture of CAR
T cells, from T cell selection through to cell
formulation, can be performed in a single-use
tubing set. This closed system greatly reduces
Figure 2: The CliniMACS Prodigy enables
complex automated cell manufacturing workflows
in a closed system.
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NEWS
GMP-compliant cell processing
in a closed system – opening up
new dimensions of scalability
Most of the current concepts for CAR T cell
therapies are based on autologous cells,
which means that each cellular product is
manufactured in a single batch in small scale
for a single patient. If the products were to
be processed in an open production line,
each single batch would almost require its
own dedicated clean room. In contrast, the
CliniMACS Prodigy, with its closed system,
enables GMP-compliant cell processing
in itself. For a future CAR T cell–based
therapy, multiple cellular products could be
manufactured in parallel in a single clean
room. This would increase the number of
cellular products that can be produced within a
specified time, and thus the number of patients
that could benefit from the therapy. Thanks
to the robust, automated, and standardized
processes, the CliniMACS Prodigy could also
enable cell manufacture close to the patient.
This would greatly simplify logistics and
avoid issues arising from transportation of the
sensitive cellular material.
The Miltenyi Biotec portfolio –
supporting the complete workflow
of cell manufacture
Besides the CliniMACS Prodigy, Miltenyi
Biotec also offers a broad range of GMPcompliant products for CAR T cell processing –
starting with the CliniMACS® Reagents for
the isolation of CD4+ or CD8+ T cells or the
isolation of T cell subsets via CD62L, the
MACS® GMP TransAct™ CD3/CD28 Kit
for the activation and expansion of T cells,
through to the TexMACS™ GMP Medium
optimized for culturing T cells, and MACS
GMP Cytokines including IL-2, IL-7, IL-15,
and IL-21. Moreover, the team from Lentigen
Technology Inc., who joined Miltenyi Biotec
in 2014, has a strong expertise in the
development and manufacturing of clinicalgrade lentiviral vectors used to genetically
engineer CAR T cells.
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The broad range of flow cytometry tools
including MACSQuant® Flow Cytometers and
hundreds of MACS Antibodies allows for a
detailed analysis of the cellular products.
With its comprehensive portfolio, Miltenyi
Biotec not only provides the basis for the
development of innovative CAR T cell–based
therapies, but also provides a strong foundation
for a future implementation of commercialscale manufacture – all with the goal to open up
the possibility of making innovative therapies
available to many patients.
References
1. Abken, H. (2015) MACS&more 16: 32–36.
2. Kalos, M. et al. (2011) Sci. Transl. Med. 3: 95ra73.
3. Maus, M. V. et al. (2014) Blood 123: 2625–2635.
4. Apel, M. et al. (2013) Chemie Ingenieur Technik
85: 103–110.
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
The CliniMACS® System components, including Reagents, Tubing
Sets, Instruments, and PBS/EDTA Buffer, are manufactured
and controlled under an ISO 13485–certified quality system.
In the EU, the CliniMACS System components are available as
CE-marked medical devices. In the US, the CliniMACS CD34
Reagent System, including the CliniMACS Plus Instrument,
CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and
LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved;
all other products of the CliniMACS Product Line are available
for use only under an approved Investigational New Drug
(IND) application or Investigational Device Exemption (IDE).
CliniMACS MicroBeads are for research use only and not for
human therapeutic or diagnostic use.
MACS® GMP Products are for research use and ex vivo cell
culture processing only, and are not intended for human in vivo
applications. For regulatory status in the USA, please contact your
local representative. MACS GMP Products are manufactured and
tested under a quality management system (ISO 13485) and are
in compliance with relevant GMP guidelines. They are designed
following the recommendations of USP <1043> on ancillary
materials. No animal- or human-derived materials were used for
manufacture of these products.
Vol 17 • 1/2016
MACS & more
5
REPORT
Dendritic cells pulsed with PepTivator® Ovalbumin
induce both OVA-specific CD4+ and CD8+ T cells
and cause antitumor effects in a mouse model
of lymphoma
Kenji Miki¹, Koji Nagaoka¹, Hermann Bohnenkamp², Takayuki Yoshimoto³, Ryuji Maekawa¹*, and Takashi Kamigaki⁴
¹ Medinet Medical Institute, MEDINET Co., Ltd., Tokyo, Japan
² Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
³ Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
⁴ Seta Clinic Group, Shin-Yokohama, Japan
* not shown
Introduction
Dendritic cell (DC)-based vaccines hold great
promise for cancer immunotherapy. DCs are
pulsed with peptides and subsequently used as
antigen-presenting cells to induce an antitumor
response in vivo through the activation of
T cells. Thus far, mostly epitope-specific
peptides with 8–10 amino acids in length have
been used to generate DC vaccines, which
however activate only CD8+ T cells. Moreover,
available single epitope-specific peptides are
restricted to MHCI or MHCII and therefore
activate either CD8+ or CD4+ T cells.
Here we used a PepTivator® Peptide Pool, which
consists mainly of 15-mer peptides covering
the complete sequence of the target antigen, in
this case ovalbumin (OVA), with an 11–amino
acid overlap. We show that DCs pulsed with
this peptide pool induced both OVA-specific
CD8+ and CD4+ T cell responses and caused
strong antitumor effects in a mouse model of
lymphoma.
Material and methods
DC generation and antigen loading
DCs were generated from mouse (C57BL/6)
bone marrow cells cultured in the presence of
miltenyibiotec.com
specific T cell receptor (TCR). CD4+ and CD8+
T cells were isolated with the CD4+ T Cell
Isolation Kit, mouse and the CD8a+ T Cell
Isolation Kit, mouse, respectively (both from
Miltenyi Biotec).
20 ng/mL GM-CSF for 10 days. Subsequently,
DCs were maturated in the presence of 10 ng/
mL GM-CSF, 10 ng/mL IL-4, and 1 µg/mL LPS.
DCs were loaded with antigen by pulsing with
PepTivator Ovalbumin peptide pool (Miltenyi
Biotec) for 4 hours. Ovalbumin (OVA) peptides
with I-Ab (MHCII)-restricted (OVA323–339) and
H-2Kb (MHCI)-restricted (OVA257–264) epitopes
were used as positive controls. All peptides
were used at the final concentration of 2 µg/mL.
Following the incubation, cells were washed
with medium to remove excess peptides.
T cell priming capacity of DCs
To test the capacity of DCs to induce T cell
proliferation, the DCs pulsed with PepTivator
Ovalbumin or the I-Ab- or H-2Kb-restricted
OVA peptides were cocultured with CFSElabeled OT-II CD4+ T cells or OT-I CD8+ T cells.
The ratio of DCs to T cells was 1:20. Untreated
DCs were used as a negative control. After
1 to 4 days of coculture, T cell proliferation was
determined by flow cytometry using absolute
T cell isolation
T cells were obtained from spleens of OT-II
or OT-I mice carrying a transgenic OVA-
• No antigen
• OVA tetramer assay
b
• S erum IgG detection
DCs • Peptide (I-A )
by ELISA
• Peptide (H-2K b)
• Peptide (I-Ab + H-2K b)
• Measurement
• PepTivator Ovalbumin
of tumor weight
E.G7
C57BL/6
0
1
2
3
4
5
6
7
8
9
10 11 12 13
14 15
Figure 1 Timeline for the evaluation of antitumor effects of DCs. Numbers indicate the days after tumor
induction with E.G7 cells.
Vol 17 • 1/2016
MACS & more
7
REPORT
A
OVA peptide–pulsed DCs induce CD4+ and
CD8+ T cells to proliferate and secrete
pro-inflammatory cytokines
Mature DCs have the capacity to induce
T cell proliferation. To test whether the OVA
peptide–pulsed DCs can exert this function, we
cocultured the DCs with CD4+ or CD8+ T cells
carrying an OVA-specific TCR and measured
the numbers of T cells after various time points.
8
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Vol 17 • 1/2016
2.0
1.0
0
1
2
3
Time (days)
5.0
3.0
2.0
1.0
peptide (I-Ab)
0
1
2
3
Time (days)
no antigen
PepTivator Ovalbumin
B
ns
4.0
0.0
4
peptide (H-2K b)
no antigen
Relative cell number
Relative cell number
no antigen
peptide (I-Ab)
PepTivator
Ovalbumin
10⁰
10¹
4
PepTivator Ovalbumin
peptide (H-2K b)
PepTivator
Ovalbumin
10³
10²
10⁰
10¹
10³
10²
CFSE
CFSE
IL-2
C
OT-II CD4 T cells
OT-I CD8+ T cells
12.0
10.0
8.0
6.0
4.0
2.0
0.0
ns
No antigen
Peptide
(I-Ab)
IL-2 level (ng/mL)
+
25.0
ns
20.0
15.0
10.0
5.0
0.0
No antigen
PepTivator
Ovalbumin
Peptide
(H-2K b)
PepTivator
Ovalbumin
IFN-γ
OT-II CD4 T cells
OT-I CD8+ T cells
+
12.0
10.0
8.0
6.0
4.0
2.0
0.0
ns
No antigen
Peptide
(I-Ab)
PepTivator
Ovalbumin
IFN-γ level (ng/mL)
Results
ns
3.0
no antigen
IL-2 level (ng/mL)
Evaluation of DC-induced immune
responses in a mouse model
On day 11, serum from three mice was
examined for the presence of OVA-specific
IgG by ELISA. Moreover, the numbers of OVAspecific CD8+ cytotoxic T cells were determined
by flow cytometry. To this end, resected tumors
were dissociated into single-cell suspensions by
treatment with collagenase. Subsequently, cells
were analyzed by flow cytometry for H-2Kb
OVA tetramer+CD8+ T cells. Cell numbers were
normalized to the weight of tumors to allow for
direct comparison of the individual mice.
5.0
4.0
0.0
IFN-γ level (ng/mL)
After 4 days, the mice were injected with 3×10⁵
DCs that were either left untreated or pulsed
with PepTivator Ovalbumin or MHCII (I-Ab)restricted or MHCI (H-2Kb)-restricted OVA
peptides or both peptides in combination.
The tumor volume was measured with a
micrometer calliper at various time points until
day 14. On day 14, six mice were sacrificed to
measure tumor size by weight.
OT-I CD8+ T cells
6.0
T cell number (×10⁵)
Assessment of antitumor effects of DCs
in a mouse model of lymphoma
The timeline for tumor induction,
immunization with DCs, and evaluation of
antitumor effects by DCs is outlined in figure 1.
Tumors were induced in C57BL/6 mice on day 0
by subcutaneously injecting 5×10⁵ E.G7-OVA
tumor cells. E.G7-OVA cells are derivatives of
EL4 mouse lymphoma cells modified to express
and secrete OVA constitutively. Therefore,
these cells can be recognized by pulsed DCs.
OT-II CD4+ T cells
6.0
T cell number (×10⁵)
count beads. Moreover, proliferation of T cells
was analyzed on day 3 by a CFSE dilution assay.
On day 2, concentrations of IL-2 and IFN-γ in
the culture supernatants were measured by
ELISA.
25.0
ns
20.0
15.0
10.0
5.0
0.0
No antigen
Peptide
(H-2K b)
PepTivator
Ovalbumin
Figure 2 Capacity of DCs to induce T cell proliferation and secretion of proinflammatory cytokines.
DCs loaded with PepTivator Ovalbumin or the MHCII (I-Ab)– or MHCI (H-2Kb)–restricted OVA peptides
were cocultured with isolated CD4+ or CD8+ T cells on day 0. T cell numbers were determined by flow
cytometry at various time points (A; n = 3; means±SD; Student’s t-test; ns: non-significant at p ≥ 0.05).
On day 3, T cell proliferation was assessed by flow cytometric CFSE dilution assay (B). Concentrations of IL-2
and IFN-γ in the supernatant of the coculture were determined on day 2 (C; n = 3; means±SD; Student’s t-test;
ns: non-significant at p ≥ 0.05).
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REPORT
A
Tumor volume (mm³)
3000
2000
*
1000
*
*
0
4
6
8
10
12
Time after tumor inoculation (days)
No antigen
Peptide (I-Ab)
14
Peptide (H-2K b)
Peptide (I-Ab + H-2K b)
PepTivator Ovalbumin
Tumor weight (mg)
B
3000
*
2000
*
*
*
1000
0
No antigen
Peptide (I-Ab)
Peptide (H-2K b)
Peptide
(I-Ab + H-2K b)
PepTivator
Ovalbumin
Figure 3 Inhibition of tumor growth by OVA-pulsed DCs. C57BL/6 mice were injected with E.G7 cells on
day 0. (A) Tumor volume was measured with a micrometer calliper at various time points after immunization
with DCs that were loaded with PepTivator Ovalbumin or the MHCII (I-Ab)– and/or MHCI (H-2Kb)–
restricted OVA peptides. (B) At day 14 after injection of tumor cells, i.e., day 10 after DC immunization,
mice were sacrificed and the tumor size was measured in weight. (A and B; n = 6; means±SD; Student’s t-test;
ns: non-significant at p ≥ 0.05; *p < 0.05.
DCs loaded with PepTivator Ovalbumin
induced an increase in the numbers of both
CD4+ and CD8+ T cells from day 2 onward,
similarly to the respective MHCII or MHCIrestricted peptides. DCs that were not loaded
with any antigen did not induce T cell
proliferation (fig. 2A). T cell proliferation was
confirmed with the CFSE dilution assay on
day 3 (fig. 2B).
Likewise, DCs loaded with PepTivator
Ovalbumin or the MHCI- or MHCII-restricted
OVA peptides induced the secretion of IL-2
and IFN-γ, in contrast to DCs that were not
loaded with antigen. Cytokine secretion was
measured on day 2 (fig. 2C).
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OVA peptide–pulsed DCs inhibit tumor
growth in vivo in a mouse model of lymphoma
Cells from the OVA-expressing mouse
lymphoma cell line E.G7 were injected into
C57BL/6 mice on day 0 to induce tumor
growth (fig. 3A). After four days the mice
were immunized by injection of DCs pulsed
with PepTivator Ovalbumin or the MHCI- or
MHCII-restricted OVA peptides, or both OVA
peptides in combination. As a negative control,
mice were injected with untreated DCs. Six
days after immunization the DCs pulsed with
OVA antigen already caused a slight decrease
in tumor size compared to the negative
control (fig. 3A). Ten days after immunization
there was a significant decrease in tumor
volume with all pulsed DCs. DCs pulsed with
PepTivator Ovalbumin or the two MHCIand MHCII-restricted OVA peptides led to
the largest reduction in tumor size by about
90%, followed by the MHCI-restricted peptide
(80% reduction). The inhibitory effect of DCs
pulsed with the MHCII-restricted peptide was
the smallest, leading to a reduction in tumor
size by about 50% (fig. 3A). Similar results were
obtained for the tumor weight ten days after
immunization (fig. 3B).
OVA peptide–pulsed DCs induce immune
responses in a mouse model of lymphoma
The tumors induced by injection of E.G7 cells
were also analyzed for OVA-specific cytotoxic
CD8+ T cells (CTLs) with a tetramer assay (fig.
4A). The numbers of CTLs were normalized
to the tumor weight, which allows for the
direct comparison of the individual mice.
DCs pulsed with PepTivator Ovalbumin or
the combination of both MHCI- and MHCIIrestricted peptides led to the highest relative
numbers of OVA-specific CTLs, whereas
the MHCI-restricted peptide resulted in a
significantly lower number of OVA-specific
CTLs. No OVA-specific CTLs were detectable
in tumors from mice treated with control DCs
or DCs pulsed with the MHCII-restricted
peptide (fig. 4A).
We also analyzed the relative titers of OVAspecific IgG in the serum. The results were
similar to the results from CTL enumeration,
except that DCs pulsed with the MHCIIrestricted peptide also induced IgG production,
just like the MHCI-restricted peptide (fig. 4B).
Conclusion
•PepTivator Ovalbumin–pulsed DCs induced
both CD4+ and CD8+ T cell responses in
vitro, similar to the MHCII- and MHCIrestricted peptides.
•
Immunization of mice bearing OVAexpressing tumors with DCs that were
previously pulsed with PepTivator
Ovalbumin or the MHCI-restricted peptide
led to infiltration of OVA-specific CD8+
CTLs into the tumor. In contrast, no CTLs
were detectable in the tumors from mice
that were treated with DCs pulsed with the
MHCII-restricted peptide. This directly
reflects the greater inhibition of tumor
Vol 17 • 1/2016
MACS & more
9
REPORT
Number of intratumor
CTLs per mg of tumor
A
150
ns
100
**
50
0
B
*
*
ns
Peptide (I-Ab)
No antigen
Peptide (H-2K b)
Peptide
(I-Ab + H-2K b)
0.8
**
0.6
OD 450
PepTivator
Ovalbumin
**
0.4
*
ns
**
0.2
0.0
No antigen
Peptide (I-Ab)
Peptide (H-2K b)
Peptide
(I-Ab + H-2K b)
PepTivator
Ovalbumin
Figure 4 Infiltration of CTLs into tumors and production of OVA-specific IgG by DCs pulsed with OVA
peptides. C57BL/6 mice were injected with E.G7 cells on day 0. On day 11, i.e., 7 days after immunization,
single-cell suspensions prepared from tumor tissue were analyzed for OVA-specific CTLs by a flow cytometric
tetramer assay (A) and the serum was tested for OVA-specific IgG by ELISA (B). CTL numbers were normalized
to tumor weight. n = 3; means ± SD; Student’s t-test; ns: non-significant at p ≥ 0.05; *0.01 ≤ p < 0.05; **p < 0.01.
growth by the DCs pulsed with PepTivator
Ovalbumin or the MHCI-restricted peptide.
•PepTivator Ovalbumin–pulsed DCs could
induce OVA-specific CD4+ T cell response
in vivo, which resulted in the efficient
production of OVA-specific IgG.
•PepTivator Ovalbumin–pulsed DCs could
induce both MHCII- and MHCI-restricted
T cell responses in vivo. Therefore, antitumor
effect and OVA-specific CTL induction by
PepTivator Ovalbumin–pulsed DCs were
stronger compared to MHCI- or MHCIIrestricted peptide-pulsed DCs.
•
Responses elicited by PepTivator
Ovalbumin-pulsed DCs may be not
restricted to MHCI or MHCII.
10
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Vol 17 • 1/2016
MACS Product
Order no.
PepTivator Ovalbumin –
research grade*
130-099-771
CD4+ T Cell Isolation Kit, mouse
130-104-454
CD8a T Cell Isolation Kit, mouse
130-104-075
Mouse GM-CSF – premium grade
130-095-739**
Mouse IL-4 – premium grade
130-097-759**
+
* For additional PepTivator Peptide Pools,
visit www.miltenyibiotec.com/peptivator
** Order numbers are provided for 100 μg sizes. For different
quality grades and additional package sizes,
visit www.miltenyibiotec.com/cytokines
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
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REPORT
Detection, enrichment, and direct phenotypic and
functional characterization of antigen-specific
naive and memory T cell subsets
Petra Bacher and Alexander Scheffold
Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany
Introduction
Infection-related mortality is a considerable
clinical challenge in immunocompromised
individuals, e.g., after hematopoietic stem cell
transplantation or chemotherapy. Pathogenspecific T cells are crucial mediators of
immune protection as shown for example by
adoptive transfer of antigen-specific T cells.
However, for both predicting or diagnosing
infectious complications as well as for the
development of effective therapies it is crucial
to have reliable methods to phenotypically and
functionally characterize the antigen-specific
T cells. In general, multicolor flow cytometry
is a robust technique to enumerate and
characterize cells according to a multitude
of parameters simultaneously. However,
antigen-specific T cells are very rare in the
naive compartment of peripheral blood
(0.2–60 cells/10⁶ naive T cells; ref. 1 and
references therein), and even in the memory
compartment their proportion is well below
1%¹. Therefore, the number of naive antigenspecific T cells, for example, is too low for the
direct ex vivo characterization by conventional
flow cytometry. To overcome these limitations,
we developed a straightforward method
for the fast and specific antigen-reactive
T cell enrichment (ARTE) based on MACS®
Technology. This technique greatly increases
the sensitivity of detection in flow cytometry,
and enables the comprehensive analysis of
extremely rare T cell subsets.² The method is
based on the immunomagnetic enrichment
of activated CD154+ or CD137+ cells. For
the detailed flow cytometric analysis of the
enriched antigen-specific T cells, we designed
comprehensive panels of fluorochromeconjugated antibodies and gating strategies.
CD154 (CD40L) is expressed specifically
on all antigen-activated conventional
CD4+ T cells upon TCR stimulation.³ We
detected CD154+ cells after stimulation of
PBMCs from healthy individuals with antigens
from Aspergillus fumigatus, Candida albicans,
cytomegalovirus (CMV), and adenovirus
(AdV) and with tetanus toxoid. For cell
enrichment and detailed characterization, we
focused on A. fumigatus–specific T cells as an
example.
Regulatory T (Treg) cells contribute to
maintaining the balance between pro- and antiinflammatory immune responses. We showed
recently that A. fumigatus causes a robust
Treg cell response in vivo⁴, counteracting
inappropriately strong immune responses in
healthy individuals. Six hours after stimulation
with antigen, Treg cells express CD137.⁵ Using
the ARTE technique to enrich CD137+ and
CD154+ cells from PBMCs stimulated with
A. fumigatus lysate in vitro, we were able
to simultaneously identify CD137–CD154+
conventional (Tcon) T cells and CD137+CD154–
Treg cells. ARTE in combination with
multicolor flow cytometry will be a valuable
tool for sensitive monitoring of antigen-specific
Naive T cell subsets
Memory T cell subsets
Cytokine-producing T cells (panel 1)
Cytokine-producing T cells (panel 2)
CD154+ Tcon and CD137+ Treg cells
CD8/14/20-VioGreen™
CD8/14/20-VioGreen
CD8/14/20-VioGreen
CD8/14/20-VioGreen
CD8/14/20-VioGreen
CD4-APC-Vio® 770
CD4-APC-Vio 770
CD4-APC-Vio 770
CD4-APC-Vio 770
CD4-APC-Vio 770
CD154-VioBlue®
CD154-VioBlue
CD154-VioBlue
CD154-VioBlue
CD154-PE-Vio 770
CD45RO-PE-Vio 770
CD45RO-PE-Vio 770
CD45RO-PE-Vio 770
CD45RO-PE-Vio 770
CD25-Brilliant Violet 421
CD197 (CCR7)-FITC
CD197 (CCR7)-FITC
Anti-TNF-α-FITC
Anti-IL-17-FITC
CD137-PE
CD45RA-APC
CD27-APC
Anti-IL-10-APC
Anti-IL-5-APC
Anti-FoxP3-APC
CD31-PE
CD95-PE
Anti-IFN-γ-PE
Anti-IL-4-PE
Anti-Helios-FITC
Table 1: Antibody panels for the analysis of naive/memory T cell subsets, cytokine-producing T cells, as well as CD154 Tcon and CD137+ Treg cells. Tandem
Signal Enhancer was added to all panels.
+
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A. fumigatus
0.00%
0.11%
C. albicans
CD154-PE
0.17%
CD154-PE
CD154-PE
w/o antigen
AdV
CMV
Tetanus
CD154-PE
0.06%
CD154-PE
0.03%
CD154-PE
0.64%
Enrichment of CD154+ and CD137+ antigenspecific T cells
After stimulation, the antigen-specific cells
were isolated using the CD154 MicroBead
Kit alone or in combination with the CD137
MicroBead Kit (both from Miltenyi Biotec)
according to the manufacturer’s instructions.
Briefly, cells were magnetically labeled with
CD154-Biotin/Anti-Biotin MicroBeads or
CD137-PE/Anti-PE MicroBeads and isolated
using two sequential MS Columns.²
CD4-APC-Vio 770
Figure 1: CD154 is a reliable marker for the detection of antigen-specific CD4+ T cells. PBMCs from
healthy donors were stimulated with A. fumigatus or C. albicans lysates, CMV or AdV peptide pools, or
tetanus toxoid for 7 h. CD154+ expression was analyzed by flow cytometry. Numbers indicate the frequencies
of antigen-reactive CD154+ cells among CD4+ T cells. Data originally published in: Bacher et al. (2013)
J. Immunol. 190: 3967–3976. Copyright © 2013 The American Association of Immunologists, Inc.
12
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CD154-VioBlue
Material and methods
Stimulation of antigen-specific T cells
PBMCs were prepared by density gradient
centrifugation from blood obtained from
healthy donors. All donors gave their informed
consent. PBMCs (1–2×10⁷ cells) from healthy
volunteers were stimulated for 7 h in RPMI
1640, supplemented with 5% AB serum, with
the following antigens: A. fumigatus lysate
(40 μg/mL, Miltenyi Biotec), C. albicans lysate
(20 μg/mL, Greer Laboratories), PepTivator®
CMV pp65, PepTivator AdV5 Hexon
(0.6 nmol/mL; both from Miltenyi Biotec),
or tetanus toxoid (10 μg/mL; Statens Serum
Institute) in the presence of CD40 pure
– functional grade and CD28 pure – functional
grade antibodies (1 μg/mL each; both from
Miltenyi Biotec). For some experiments, cells
were stained intracellularly with anti-cytokine
antibodies. In this case, 1 μg/mL of brefeldin A
was added to the cells for the last two hours of
stimulation.²
No enrichment
w/o antigen
0.02%
20
A. fumigatus
0.00%
0
0.06%
70
0.04%
46
Anti-TNF-α-PE-Vio 770
Enriched fraction
w/o antigen
CD154-VioBlue
T cells in various important immune-mediated
diseases, such as autoimmunity, inflammatory
bowel disease, allergy, and tumor immunology
as well as for the development of specific
immunotherapies.
Cell staining and flow cytometry
Cells were stained for multiparametric
flow cytometry with different panels of
fluorochrome-conjugated antibodies (table 1),
depending on the subset to be analyzed. All
antibodies were from Miltenyi Biotec except
CD25-Brilliant Violet™ 421 (BioLegend®).
For flow cytometry analysis of cytokinesecreting antigen-specific T cells, the
stimulated cells were labeled fluorescently
during the enrichment procedure: Cells labeled
with MACS MicroBeads were applied to the
first MS Column and subsequently stained
5.03%
71
1.22%
18
A. fumigatus
14.4%
821
48.9%
2791
Anti-TNF-α-PE-Vio 770
Figure 2: Enrichment of CD154+ cells allows for highly sensitive enumeration and characterization of
rare antigen-specific CD4+ T cells. PBMCs from a healthy donor were stimulated with A. fumigatus lysate or
left unstimulated. CD154 expression was assessed among CD4+ T cells without prior enrichment (upper plots)
and after CD154+ cell enrichment (lower plots). Bold numbers indicate the total count of CD154+ cells after
acquiring 4×10⁵ PBMCs (upper plots) or the enriched fraction obtained from 1.5×10⁷ PBMCs (lower plots).
miltenyibiotec.com
REPORT
CD45RO-PE-Vio 770
22.3%
61.9%
CD197 (CCR7)-FITC
31.2%
22.7%
RTE
CD31-PE
Gated on
CD45RO– CCR7+
15.2% Tcm
54.7% Tn
0.2% Tscm
CD95-PE
4.7%
Tem
CD197 (CCR7)-FITC
Figure 3: Characterization of naive and memory T cell subsets. A. fumigatus–specific CD154+ T cells were
enriched as described and counterstained for phenotypic markers to discriminate between naive and memory
T cell subsets. Percentages of the respective cell subsets among all reactive CD154+ T cells are shown. RTE:
recent thymic emigrants; CD31– naive cells: peripheral circulating naive T cells; Tn: naive T cells; Tscm: stem
cell memory T cells; Tcm: central memory T cells; Tem: effector memory T cells
Phenotypic characterization of antigenspecific naive and memory subsets
To further dissect the enriched CD154 +
A. fumigatus–specific T cell population by flow
cytometry, we designed two antibody panels
(see material and methods section). The gating
Highly sensitive enumeration and
strategy illustrated in figure 3 allowed us to
characterization of rare antigen-specific
easily distinguish naive from memory T cells.
CD4+ T cell subsets
To enable the sensitive analysis of rare antigen- Moreover, we were able to determine the
specific T cell subsets by flow cytometry, we proportions of recent thymic emigrants (RTE,
magnetically enriched the activated CD154+ 22.7% of CD154+ cells), peripheral circulating
cells after stimulation with A. fumigatus lysate. naive T cells (31.2%), stem cell memory T cells
In the example shown in figure 2, only about (Tscm; 0.2%), central memory T cells (Tcm;
120 CD154+ cells were detected after acquiring 15.2%), and effector memory T cells (Tem;
4×10⁵ PBMCs. In contrast, after enrichment 4.7%). These data show that the possibility of
of CD154+ cells from 1.5×10⁷ PBMCs and measuring a large number of rare target cells
acquisition of the entire positive fraction, more pre-enriched from a large blood sample greatly
than 3,600 CD154+ cells were detected among improves the significance of the multiparameter
only approx. 5×10⁴ total events, whereas approach, permitting identification of small
background levels in the nonstimulated sample cell subsets at high resolution. Small subsets,
remained low (<100 cells). These results such as Tscm, would not be detectable without
indicate that enrichment of CD154+ T cells prior enrichment of the antigen-specific T cells.
prior to flow cytometry greatly increases the
signal-to-noise ratio for a sensitive analysis of
antigen-specific T cell subsets.
miltenyibiotec.com
Gated on
CD45RO+
CD27-APC
Gated on
CD45RO– CCR7+
CD197 (CCR7)-FITC
Results
Detection of antigen-specific CD4+CD154+
T cells
The CD154 antigen is a reliable marker for
the detection of activated antigen-specific
T cells.³ We first determined the proportion
of CD154+ T cells in PBMCs from healthy
donors, upon stimulation with antigens from
A. fumigatus, C. albicans, CMV, and AdV and
with tetanus toxoid for 7 h. The percentage of
the entire population of activated CD154+ cells
among CD4+ cells could be determined reliably
(range: 0.03%–0.64%; fig. 1). However, the total
number of CD154+ cells in the samples was too
low to characterize smaller subpopulations,
such as naive and memory T cells, by flow
cytometry.
CD4 +CD154 +
CD45RA-APC
on the column with fluorochrome-conjugated
antibodies. Cells were eluted for fixation (Inside
Stain Kit, Miltenyi Biotec) and subsequently
applied to the second MS Column, where
they were permeabilized (Inside Stain Kit,
Miltenyi Biotec). Intracellular cytokines were
stained while the cells were still on the column.
The transcription factors FoxP3 and Helios
were stained using the respective antibodies
in combination with the FoxP3 Staining Buffer
Set (Miltenyi Biotec). After elution from the
second column, the cells were analyzed by flow
cytometry on a MACSQuant® Analyzer 10
with the MACSQuantify™ Software (both from
Miltenyi Biotec).²
Characterization of cytokine production
in antigen-specific naive and memory
T cells
The high resolution is also important for the
analysis of cytokine-producing subsets. We
compared the cytokine production capacity of
the naive and memory T cell subsets by flow
cytometry, based on two antibody panels (see
material and methods section). These panels
enabled us to determine the percentages of cell
subsets producing TNF-α, IFN-γ, IL-10, IL-17,
IL-4, or IL-5. In the example shown in figure 4,
the majority (70.4%) of naive CD4+CD154+
T cells produced TNF-α, whereas effector
cytokines were almost absent (<1%). Likewise,
the majority (63.6%) of memory CD4+CD154+
T cells produced TNF-α. More than 10% of the
memory subset produced IFN-γ, indicating the
presence of Th1 cells. However, only small but
still significant proportions of memory but not
naive T cells produced IL-10, IL-17, or IL-4.
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MACS & more
13
REPORT
Gated on naive CD4 + T cells
CD154-VioBlue
70.4%
Anti-TNF-α-FITC
0.6%
Anti-IFN-γ-PE
0.4%
Anti-IL-10-APC
0.1%
0.1%
Anti-IL-17-FITC
0.1%
Anti-IL-4-PE
Anti-IL-5-APC
Gated on memory CD4 + T cells
CD154-VioBlue
63.6%
Anti-TNF-α-FITC
11.2%
Anti-IFN-γ-PE
4.1%
2.7%
Anti-IL-10-APC
Anti-IL-17-FITC
3.4%
0.3%
Anti-IL-4-PE
Anti-IL-5-APC
Figure 4: Characterization of cytokine production in antigen-specific naive and memory T cells. A. fumigatus-specific CD154+ T cells were enriched by ARTE
and analyzed for cytokine expression within the antigen-specific naive and memory compartments. Cells were gated on CD4+ lymphocytes. Percentages of cytokineexpressing cells among CD154+ T cells are shown.
2665
1.1%
Anti-FoxP3-APC
2404
1%
0.1%
Anti- Helios-FITC
CD137-PE
86.6%
Anti-FoxP3-APC
Anti-FoxP3-APC
Gated on CD137+ T cells
CD25-BV421
CD154-PE-Vio 770
Gated on CD4 + T cells
Anti-FoxP3-APC
CD25-BV421
Gated on CD154 + T cells
29.7%
56.9%
Anti-Helios-FITC
Gated on CD4 + T cells
CD137-PE
CD154-PE-Vio 770
Enrichment of CD154+ and CD137+ cells
enables identification of antigen-specific
Tcon and Treg cells in parallel
Using the ARTE technique based on the
expression of CD154 and CD137⁴,⁵, we were
able to differentiate between CD137–CD154+
Tcon and CD137+CD154– Treg cells⁶. Almost
all CD137+ cells co-expressed FoxP3, whereas
FoxP3 expression was absent in CD154+ cells
(fig. 5, lower dot plots). Around 90% of the
CD137+ cells were positive for FoxP3 and
CD25, and the majority of FoxP3+ cells coexpressed the transcription factor Helios.
In contrast, CD154+ T cells had a conventional
T cell phenotype (CD25 –FoxP3 –Helios –).
These data are in line with our findings that
A. fumigatus induces a significant Treg response
in vivo⁴, which can control excessive immune
responses, such as allergies.
Anti-FoxP3-APC
Anti-FoxP3-APC
Figure 5: Combined enrichment of CD154+ and CD137+ cells enables parallel identification of antigenspecific Tcon and Treg cells. PBMCs were stimulated with A. fumigatus lysate and reactive CD154+ and
CD137+ T cells were enriched by ARTE. Enriched CD154+ and CD137+ were counterstained for typical Treg
cell markers CD25, FoxP3, and Helios.
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Vol 17 • 1/2016
miltenyibiotec.com
REPORT
Conclusion
•
Enrichment of antigen-reactive T cells,
based on MACS Technology, enhances the
signal-to-noise ratio for sensitive multicolor
flow cytometry.
•The collection of large numbers of rare
target cells following magnetic preenrichment greatly improves the resolution
of downstream multiparameter flow
cytometric analyses.
•
C omprehensive panels of MACS
Antibodies enable the detailed phenotypic
characterization of the enriched antigenspecific CD154+ T cells for the distinction
between naive and memory T cell subsets as
well as the analysis of cytokine production
in naive and memory T cells.
•The parallel enrichment of CD137+ and
CD154+ cells and a specific antibody panel
allow for the characterization of CD137–
CD154+ Tcon cells and CD137+CD154–
Treg cells.
References
1. Bacher, P. and Scheffold, A. (2013) Cytometry A
83: 692–701.
2. Bacher, P. et al. (2013) J. Immunol. 190:
3967–3976.
3. Frentsch, M. et al. (2005) Nat. Med. 11:
1118–1124.
4. Bacher, P. et al. (2014) Mucosal Immunol. 7:
916–928.
5. Schoenbrunn, A. et al. (2012) J. Immunol. 189:
5985–5994.
6. Bacher, P. et al. (2014) J. Immunol. 193:
3332–3343.
MACS Pr oduct
Order no.
Cell isolation
CD154 MicroBead Kit, human
130-092-658
CD137 MicroBead Kit, human
130-093-476
Cell culture and stimulation
A. fumigatus Lysate
130-098-170
PepTivator CMV pp65, human
130-093-435
PepTivator AdV5 Hexon
130-093-496
CD28 pure – functional grade,
human
130-093-375
CD40 pure – functional grade,
human
130-094-133
Flow cytometry
MACSQuant Analyzer 10
130-096-343
MACSQuantify Software
130-094-556
Anti-FoxP3-APC, human and
mouse (clone: 3G3)
130-093-013
Anti-Helios-FITC, human and
mouse (clone: 22F6)
130-104-000
Anti-IFN-γ-PE (clone: 45-15)
130-091-653
Anti-IL-4-PE (clone: 7A3-3)
130-091-647
Anti-IL-5-APC
(clone: JES1-39D10)
130-091-834
Anti-IL-10-APC (clone: JES3-9D7)
130-096-042
Anti-IL-17-FITC (clone: CZ8-23G1) 130-094-520
Anti-TNF-α-FITC (clone: cA2)
130-091-650
CD4-APC-Vio 770 (clone: VIT4)
130-096-652
CD8-VioGreen
(clone: BW135/80)
130-096-902
CD14-VioGreen (clone: TÜK4)
130-096-875
CD20-VioGreen (clone: LT20)
130-096-904
CD27-APC (clone: LG.3A10)
130-097-218
CD31-PE (clone: AC128)
130-092-653
CD45RA-APC (clone: T6D11)
130-092-249
CD45RO-PE-Vio 770
(clone: UCHL1)
130-096-739
CD95-PE (clone: DX2)
130-092-416
CD137-PE (clone: 4B4-1)
130-093-475
CD154-VioBlue (clone: 5C8)
130-096-217
CD154-PE-Vio 770 (clone: 5C8)
130-096-793
CD197 (CCR7)-FITC, human
(clone: REA108)
130-099-172
FoxP3 Staining Buffer Set
130-093-142
Inside Stain Kit
130-090-477
Tandem Signal Enhancer,
human
130-099-888
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
miltenyibiotec.com
Vol 17 • 1/2016
MACS & more
15
gentleMACS™ Octo Dissociator
with Heaters
Start smart with automated tissue dissociation
Multiple sample processing
Dissociation or homogenization of up to eight
samples in one go, controlled independently
Walk-away tissue dissociation
Integrated heaters for on-instrument
enzymatic incubation
Flexibility
Applicable to virtually any tissue using pre-set
or user-defined programs
Reliability
Fully automated, standardized procedures
for highly reproducible results
Time-saving
Minimal hands-on time, short procedures
Safety
Sample processing in a closed,
sterile system
miltenyibiotec.com/gentlemacs
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
REPORT
Evaluation of metastatic burden and recovery
of human metastatic cells from a mouse model
Lorena Landuzzi¹, Arianna Palladini², Marianna Lucia Ianzano², Roberta Laranga², Giulia D’Intino³, Patrizia Nanni², and Pierluigi Lollini²
¹ Laboratory of Experimental Oncology, Rizzoli Orthopedic Institute, Bologna, Italy
² Laboratory of Immunology and Biology of Metastasis, Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Bologna, Italy
³ Miltenyi Biotec S.r.l., Calderara di Reno, Bologna, Italy
Here we used the Mouse Cell Depletion Kit (human breast carcinoma cell line expressing
Introduction
Metastatic dissemination is the major cause (Miltenyi Biotec) to enrich and quantify enhanced green fluorescent protein, EGFP)1
of death in cancer. Xenotransplantation of metastatic human cells, derived from human were added. The brain tissues, spiked with
tumor tissue into immunodeficient mice is a breast carcinoma, in a model of brain different amounts of MDA-MB-453 EGFP
widespread preclinical technique to study tumor metastases1 in NOD scid gamma (NOD.Cg- cells, were dissociated enzymatically and
mechanically into single-cell suspensions,
development. However, preclinical studies on the Prkdc scid Il2rg tm1Wjl/SzJ, NSG) mice.
using the Tumor Dissociation Kit, human
spreading of metastases were so far hampered
(5 mL/sample) and the gentleMACS™ Octo
by the poor dissemination of malignant human Material and methods
Dissociator with Heaters (both from Miltenyi
tumors in conventional immunodeficient hosts, Ethics statement
like the nude mouse. The development of highly All animal experiments were performed Biotec) with program “37_h_TDK_1”. After
immunodeficient knockout mice spurred a according to the European directive dissociation each sample was passed through
new wave of metastatic model systems. It was 2010/63/UE and Italian law (DLGS 26/2014). a MACS® SmartStrainer (pore size 70 μm) and
recently shown that human HER-2-positive Experimental protocols were reviewed and split into two equal portions, one of which was
breast cancer cells, which do not metastasize approved by the Institutional Animal Care and used for flow cytometry analysis. The other half
in nude mice, when implanted in knockout Use Committee (“Comitato Etico Scientifico was subjected to treatment with the Mouse Cell
mice with severe immunodeficiency, give rise per la Sperimentazione Animale”) of the Depletion Kit from Miltenyi Biotec (20 µL of
to multiorgan metastatic patterns resembling University of Bologna, and forwarded to the MicroBeads per sample), which enables the
immunomagnetic removal of mouse cells from
those observed in human patients1. The growth Italian Ministry of Health.
the cell suspension. Subsequently, the samples
of metastatic nodules in a variety of locations,
including brain, lungs, liver, kidneys, adrenals, Recovery of human tumor cells spiked into were counted in a Neubauer hemocytometer
under a fluorescence microscope and used
ovaries, and bone marrow, opens up the problem dissected mouse brain tissue
of quantifying metastatic burden and recovering Mouse brain was cut into small pieces and split for flow cytometry analysis. HER-2 detection
human metastatic cells from mouse organs2 into four equal portions. To each portion, 0 or was performed by means of the PE-antifor cellular and molecular studies in vitro.
0.5×10⁶ or 1×10⁶ MDA-MB-453 EGFP cells huHER-2 monoclonal antibody, clone Neu 24.7
(BD® Biosciences).
Mouse tissue
Number of MDA-MB-453
EGFP cells added
Number of human EGFP+ tumor cells after
dissociation and depletion of mouse cells
Brain (one eighth)
0.5×10⁶
0.315×10⁶ (recovery 63%)
Brain (one eighth)
0.25×10⁶
0.165×10⁶ (recovery 66%)
Table 1: Recovery of MDA-MB-453 EGFP cells added to dissected mouse brain. MDA-MB-453 cells
were added to dissected brain in the indicated proportions. Subsequently, the mixtures were dissociated,
and the resulting cell suspensions were depleted of mouse cells. Numbers of EGFP+ human tumor cells were
determined microscopically. The recovery of EGFP+ cells (percentage in relation to input cells) is indicated
in parentheses.
miltenyibiotec.com
Induction of metastases in the mouse model
For the induction of metastasis 2×10⁶ MDAMB-453 EGFP cells were injected i.v. into two
NSG mice1. After approximately 2 months the
mice were sacrificed and subjected to accurate
necropsy1. The whole mice were analyzed
by fluorescence imaging for the presence of
EGFP+ cells1.
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A
18
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Vol 17 • 1/2016
RelativeEvents
cell number
Relative
cell number
Events
MDA-MB-453 EGFP cells only,
before mixing
Brain + MDA-MB-453 EGFP cells,
after dissociation
D
Brain + MDA-MB-453 EGFP cells,
after dissociation and depletion
RelativeEvents
cell number
C
5% human EGFP+
cells
FL1
EGFP
74% human
EGFP+ cells
FL1
EGFP
100% human
HER-2+ cells
HER-2
HER-2PE
F
Brain + MDA-MB-453 EGFP cells,
after dissociation and depletion
RelativeEvents
cell number
MDA-MB-453 EGFP cells only,
before mixing
Relative
cell number
Events
E
100% human
EGFP+ cells
FL1
EGFP
FL1
EGFP
Results
Effective separation of human tumor cells
from mouse brain suspensions
Xenotransplantation of human tumor tissue is a
powerful technique to mimic tumors in mouse
models. The tumor-bearing tissue is dissected
and the xenograft-derived cells can be analyzed
on a cellular or molecular level3-⁵. However,
contaminating mouse cells can severely hamper
the analysis. In order to avoid erroneous results,
it is therefore highly desirable to purify the
human cells prior to analysis.
We used the Tumor Dissociation Kit, human
and the gentleMACS Octo Dissociator with
Heaters to dissociate mouse brain tissue with
disseminated human tumor cells. The Mouse
Cell Depletion Kit was used to enrich the
human cells subsequently.
In a first experiment, we tested whether the
experimental setup would enable us to reenrich EGFP-expressing human tumor cells
that were spiked into dissected mouse brain.
The purity of EGFP+ cells after depletion of
mouse cells amounted to 74% (fig. 1D). Usually,
more than 60% of the input tumor cells were
recovered (table 1), indicating that the system
should also enable us to recover xenografts
from mouse models.
We also took the opportunity to analyze
the surface expression of HER-2, the
characteristical oncogene/antigene of these
breast cancer cells⁴. Comparison of panels E
and F in figure 1 shows that HER-2 expression
and detection were not altered by enzymatic/
mechanical tissue dissociation and mouse
cell depletion.
B
Brain only, before mixing
RelativeEvents
cell number
Recovery of human tumor cells from
disseminated metastases in mouse brain
The brains were dissected and subsequently
dissociated using the Tumor Dissociation Kit,
human (5 mL/sample) and the gentleMACS
Octo Dissociator with Heaters (program
“37_h_TDK_1”). After dissociation each
sample was split into two portions. One fifth
of the brain was used for cell counting and flow
cytometry1, and 4/5 of the brain were subjected
to treatment with the Mouse Cell Depletion Kit
(100 µL of MicroBeads per brain sample). After
the depletion of mouse cells, the samples were
counted in a Neubauer hemocytometer under
a fluorescence microscope and used for flow
cytometry analysis.
74% human
HER-2+ cells
HER-2
PE
HER-2
Figure 1: Recovery of MDA-MB-453 EGFP cells added to dissected mouse brain tissue. Flow cytometry
analysis of cell suspensions of dissociated brain tissue alone (A), MDA-MB-453 EGFP cells alone (B), mixtures
of brain tissue (one eighth of total brain) and 0.5×10⁶ MDA-MB-453 EGFP cells after dissociation (C) and after
depletion of mouse cells (D). HER-2 expression in MDA-MB-453 cells before mixing (E) and after mixing with
mouse brain tissue followed by dissociation and mouse cell depletion (F). Numbers indicate the percentage
of EGFP+ cells and HER-2+ cells. In panel E, the open profile represents MDA-MB-453 cells incubated with
PE-control isotype antibody.
Mouse tissue
Number of human EGFP+
tumor cells after tissue
dissociation
Number of human EGFP+ tumor cells after
dissociation and depletion of mouse cells
Brain metastases (mouse 1)
9.64×10⁶
5.9×10⁶ (recovery: 61%)
Brain metastases (mouse 2)
11.24×10⁶
6.94×10⁶ (recovery: 61%)
Table 2: Recovery of human metastatic cells from dissociated mouse brains. Dissected mouse brains were
dissociated into single-cell suspensions and subsequently depleted of mouse cells. Numbers of EGFP+ human
tumor cells were determined microscopically after each procedure The recovery of EGFP+ cells after depletion,
i.e., the percentage in relation to EGFP+ cells present before depletion, is indicated in parentheses.
miltenyibiotec.com
REPORT
A
Mouse 1, brain after dissociation
Relative cell number
Events
Side scatter
Side scatter
256
0
0
Forward scatter
21% human
EGFP+ cells
FL1
256
EGFP
FSC
Mouse 2, brain after dissociation
Relative cell number
Events
Side scatter
Side scatter
256
0
0
Forward scatter
256
Conclusion
17% human
EGFP+ cells
FL1
EGFP
FSC
B
Mouse 1, brain after dissociation and mouse cell depletion
Relative cell number
Events
Side scatter
Side scatter
256
250
0
0
Forward scatter
256
84% human
EGFP+ cells
FL1
EGFP
FSC
Mouse 2, brain after dissociation and mouse cell depletion
Relative cell number
Events
Side scatter
Side scatter
256
0
0
Forward scatter
256
92% human
EGFP+ cells
FL1
EGFP
FSC
Figure 2: Flow cytometry analysis of EGFP+ tumor cells in cell suspensions from two metastasesbearing mouse brains. After dissection the metastases-bearing brains were enzymatically and mechanically
dissociated, and the resulting cell suspensions were depleted of mouse cells. Cells were analyzed after tissue
dissociation (A) and after mouse cell depletion (B). Forward scatter and side scatter plots of each sample are
shown with gates used to analyze the percentage of EGFP+ cells. Histograms show the percentage of EGFP+
human tumor cells.
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Recovery of human metastatic cells from
mouse brain metastases
We used i.v. injection of MDA-MB-453 EGFP
cells into NSG mice to induce metastases1.
After approximately 2 months, heavy metastatic
burden was observed in the brains of the NSG
mice. Using the same basic experimental
setup as above, we were able to enrich the
human tumor cells effectively. Cells derived
from metastases in the brain were enriched
to a purity of 84–92% (fig. 2B). The recovery
of tumor cells, i.e., the percentage in relation
to tumor cells present before depletion of
mouse cells, was greater than 60% (table 2)
and proportional to the tumor cell burden in
the brain.
The combination of enzymatic and mechanical
tumor dissociation and subsequent depletion
of mouse cells enabled us to both quantitate
and enrich human tumor cells from brain
metastases grown in immunodeficient mice.
This is a promising system particularly for the
quantification of the metastatic burden in the
brain and other organs of mice bearing human
tumors. Simultaneously, the human metastatic
cells can be enriched for in vitro studies by
removing mouse cells. This could be of interest
to all researchers involved in the preclinical
development of new antimetastatic drugs.
References
1. Nanni, P. et al. (2012) PLoS One. 7: e39626.
2. Nanni, P. et al. (2010) Eur. J. Cancer 46: 659–668.
3. De Giovanni, C. et al. (2012) Br. J. Cancer 107:
1302–1309.
4. Menotti, L. et al. (2009) Proc. Natl. Acad. Sci. USA
106: 9039–9044.
5. Nanni, P. et al. (2013) PLoS Pathog. 9:e1003155.
Acknowledgments
M.L.I. and A.P. are in receipt of a Postdoctoral Fellowship from
University of Bologna. R.L. is in receipt of a Ph.D. Fellowship
from University of Bologna. The research of the authors is
supported by the Italian Association for Cancer Research, AIRC,
project n. 15324, Milan, Italy.
MACS Product
Order no.
gentleMACS Octo
Dissociator with Heaters
130-096-427
Tumor Dissociation Kit, human
130-095-929
Mouse Cell Depletion Kit
130-104-694
MACS SmartStrainers (70 μm)
130-098-462
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
Vol 17 • 1/2016
MACS & more
19
PERSPECTIVES
Designing a dendritic cell–based therapy
for primary liver cancer
Stuart M. Curbishley, Miroslava Blahova, and David H. Adams
University of Birmingham Medical School, National Institute for Health Research (NIHR)
Birmingham Liver Biomedical Research Unit and Centre for Liver Research, Birmingham, UK
Hepatocellular carcinoma –
an introduction
Hepatocellular carcinoma (HCC) is the most
common primary hepatic tumour and one of
the most prevalent cancers worldwide. It is
especially common in Asia and Sub-Saharan
Africa. Risk factors for its development
worldwide include hepatitis B, hepatitis C,
alcohol abuse, aflatoxin exposure, and
metabolic liver disease.
The incidence of HCC is rising, both in the
United States and the United Kingdom, and
is likely to be a reflection of the increased
prevalence of cirrhosis from three main causes:
hepatitis C, fatty liver disease, and alcoholic
liver disease (ALD). It is therefore considered
to become a major health burden in the UK in
the coming years.
Current treatment for HCC is limited and
5-year survival for all stages combined is less
than 5%. At present, surgery (either tumour
resection or liver transplantation) is the only
potentially curative treatment for HCC.
However, resection is only feasible in less than
10% of patients, as it requires small tumours,
limited-stage disease, and good hepatic
function. Hence, the majority of patients
present with advanced disease that is deemed
unresectable. The survival rate is relatively
poor, even in those patients who undergo
surgical resection, with high recurrence rates
and a 5-year survival of about 30–60%.
20
MACS & more
Vol 17 • 1/2016
of tumours of various types. For instance alphafetoprotein (AFP) is a serum marker for HCC.
For patients with unresectable but localised
HCC, local ablative therapy can remain an
option. However, for those patients who are
unsuitable for surgery or local ablation but with
liver-confined disease, palliative benefit may be
gained from transarterial chemoembolisation
(TACE). Two randomised, controlled
trials have shown that TACE performed
with doxorubicin¹ or cisplatin² improves
survival in selected patients compared to
best supportive care. Even so, TACE remains
palliative and disease progression is inevitable,
such that combination with novel therapies
is attractive. Since TACE may liberate an
abundance of tumour antigens it may lend
itself to combination with immunotherapeutic
strategies.
Proteins that are specifically or predominantly
expressed by tumours are potential targets
for immunotherapy. Standard vaccination
strategies have had only limited success in
stimulating anti-tumour immunity, leading to
the use of adoptive therapy with T cells or
dendritic cells (DCs) to stimulate more potent
immune responses which selectively kill
malignant cells expressing specific antigens.
Tumour antigens expressed in varying degrees
in HCC and thus potential targets for
immunotherapy include AFP, MAGE-1 and
3, SSX-1 and 4, TSPY, NY-ESO-1, and
Glypican-3.³–⁵
The immunotherapy
of malignancy: antigens
The immunotherapy
of malignancy: DCs
As immune-mediated mechanisms play an
important role in controlling the growth of
some types of cancer, immunotherapy could in
theory exploit such responses to generate
therapeutic immune responses against tumour
antigens. Many tumours have altered gene
regulation resulting in expression of specific
tumour antigens or overexpression of other
proteins. One such category is the cancer/testis
antigens, a category of tumour antigens that are
normally expressed in male germ cells but not
in adult somatic tissues. In malignancy, this
gene regulation is disrupted, resulting in
cancer/testis antigen expression in a proportion
DCs are potent antigen-presenting cells which
exist in peripheral tissues where they take up
and process antigens. Short peptide fragments
of the antigens are then presented on the
cell surface in association with the major
histocompatibility complex (MHC). Signals
released by infected or damaged tissues act
through receptors on the DCs to promote
their activation and maturation. Maturation
is associated with expression of costimulatory
molecules that allow the DCs to activate T cells
and chemokine receptors that mediate DC
migration from peripheral tissue into draining
lymph nodes. In the lymph nodes DCs interact
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PERSPECTIVES
with and activate T cells that recognise the
presented epitopes, resulting in the generation
of effector T cells that can mount antigenspecific anti-tumour immune responses.
Following the demonstration that activated
DCs were potent inducers of immune responses
when adoptively transferred into animals, the
development of techniques to generate large
numbers of monocyte-derived DCs (Mo-DCs)
from peripheral blood enabled DC-based
immunotherapy to be tested in clinical trials.
We previously conducted a clinical trial⁶ to
assess the safety and efficacy of intravenous
vaccination with mature Mo-DCs, pulsed
ex vivo with a liver tumour cell line lysate
(HepG2), in patients with advanced HCC.
Furthermore, we have conducted tracking
studies to assess the differential homing of MoDCs delivered intravenously or directly into
the liver via the hepatic artery (manuscript in
preparation). These studies demonstrated that
autologous Mo-DC vaccination in patients
with HCC is safe and well tolerated and that
increased numbers of Mo-DCs can be retained
in the liver when infused via the hepatic artery.
Developing the next generation
of Mo-DC vaccines in Birmingham
Following the successful completion
of our previous clinical trials involving
Mo-DCs, we looked to develop a new
study to extend our understanding of these
vaccines in patients with HCC. This led to the
development of the ImmunoTACE clinical
trial (long title: A randomised phase II clinical
trial of conditioning cyclophosphamide
and chemoembolisation with or without
vaccination with dendritic cells pulsed
with HepG2 lysate ex vivo in patients with
Hepatocellular Carcinoma; EUDRACT # 2011001690-62). This would combine the standardof-care TACE with a Mo-DC vaccination and
the addition of preconditioning in all patients
with cyclophosphamide, which at low doses has
been shown to selectively deplete regulatory
T cells and increase antigen-specific immune
responses in patients with HCC⁷.
Changes to the regulatory landscape between
the closure of our first HCC Mo-DC trial and
the development of ImmunoTACE saw the
introduction of new guidelines concerning
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the manufacture of Advanced Therapeutic
Medicinal Products (ATMP). In order to be
compliant with these current regulations it was
necessary for us to develop our manufacturing
process into a “closed” system.
Cell enrichment and culture process
The initial phase of this work included the use
of CD14 MicroBeads from Miltenyi Biotec
to isolate monocytes from peripheral blood
samples. The CliniMACS® System provides a
GMP-compliant platform for the isolation of
CD14 monocytes, typically from leukocyterich apheresis. Based around a single-use,
closed tubing set, the relative cost of CD14+
cell enrichment using this method is expensive
and clearly would not be cost effective when
beginning a new process development. With
this in mind, we chose to begin the definition
of our manufacturing process using CD14+
monocytes that were enriched from single
units of whole blood with Miltenyi Biotec’s
autoMACS® Pro Separator and GMP-grade
CliniMACS MicroBeads. The introduction of
this automated step removed any operator bias
in the preparation of the starting monocyte
population before differentiation into
Mo-DCs. As with many previous trials
involving Mo-DCs, we chose to differentiate
monocytes in the presence of GM-CSF and
IL-4 (both 1,000 IU/mL). Here, we used
premium-grade cytokines from Miltenyi
Biotec which, whilst not certified as GMP
grade, are prepared to the same standard and,
importantly, are released with a lot-specific
activity enabling us to ensure consistent dosing
of cells in all experiments.
The next stage of our process development
required transfer of our cell culture from
traditional multi-well plates into a closed bag
system. We compared culture bags and media
from several manufacturers and found little
variability in the quality of Mo-DCs prepared
in each. Further development runs were
performed in DendriMACS™ GMP Medium
(Miltenyi Biotec). Usually a limitation of
bag culture vessels is the need for a relatively
high medium volume and consequently the
requirement of high initial cell numbers. As an
alternative to single-chamber bag systems we
performed our initial culture experiments in
Miltenyi Biotec’s MACS GMP Cell Expansion
Bags. These have the advantage of multiple
chambers that can be used flexibly, according
to the required medium volume. This enabled
us to begin process development with a bag
material that is easily scalable to the next level.
The initial monocyte number was set to 3×10⁶
to account for medium additions on days
3 and 5.
On day 6 of culture the resulting immature
DCs were loaded with antigen (see below)
and matured in the presence of the TLR4
ligand monophosphoryl lipid A (MPLA). This
maturation step was chosen as the material
is available as a cGMP product and a single
maturation agent lends itself to a simpler
process with fewer interventions and is more
cost effective. Furthermore, we have previously
demonstrated that maturation via TLR4 ligation
leads to potent up-regulation of costimulatory
molecules as well as the lymph node homing
receptor CCR7. This final maturation and
antigen-loading phase continued for 48 hours
until Mo-DCs are released on day 8.
Preparation of antigen
The choice of antigen to be loaded onto
Mo-DC vaccines has long been a source of
much discussion because tumour antigens
are often shared self-antigens and thus elicit
a tolerogenic response. As our understanding
of tolerance within the immune system
increases, the combination of inhibitors of
checkpoint molecules such as CTLA-4 and
PD-1/PD-L1⁸,⁹ and perturbation of regulatory
cells has led to a resurrection of host tumour
as an antigen source. The ImmunoTACE
clinical trial was developed ahead of these
exciting new changes. For this reason, and
for pragmatic reasons associated with access
to host tumour, we decided to use a cancer
cell line lysate as a source of antigen. In our
case, the hepatoblastoma cell line HepG2 was
chosen, partly due to our previous vaccine trial
experience and because this cell line shares
some antigens associated with HCC.
Typically, preparation of lysates for clinical
application makes use of repeated freeze/
thaw cycles to induce cell lysis. This method,
whilst simple, often leads to low total protein
yields and can result in residual whole-cell
contamination. We again chose to make
Vol 17 • 1/2016
MACS & more
21
PERSPECTIVES
use of a frequently used tool in our research
laboratory from the Miltenyi Biotec stable. We
have used the gentleMACS™ Dissociator for
many years to generate single-cell suspensions
from whole tissue and to prepare material for
molecular biology protocols. Here, we used
gentleMACS M Tubes with the RNA_01.01
protocol to generate a lysate for clinical use.
Briefly, HepG2 cells were harvested from
Corning® CellStack® – 5 Chambers using
a closed bag system (Macopharma) and
cGMP trypsin substitute (TrypLE™ Select,
Invitrogen™) before being washed in PBS and
resuspended in normal saline supplemented
with calcium and magnesium. Individually
wrapped sterile gentleMACS M Tubes were
taken into a grade A processing area and
7 mL of the HepG2 cell suspension (approx.
35×10⁶ cells) were added via injection through
the septa to keep the process closed. The lids
were then firmly closed and each tube processed
on a gentleMACS Dissociator using protocol
RNA_01.01, repeated 5 times. Following lysis,
500 IU DNAse I (Pulmozyme®, Roche) was
added and the tubes incubated at 37 °C for
10 minutes. The lysate was then recovered
using a long needle and syringe via the septa
and transferred to a sterile bag and washed in
normal saline. Following final resuspension
in saline the lysate was aliquoted into 2 mL
crimp-sealed vials and stored at –80 °C.
A representative sample of the batch produced
was removed and tested for sterility, endotoxin
contamination, and presence of whole-cell
contamination before being released for use in
the clinical manufacturing process.
Final release and cryopreservation
Choosing a suitable assay to define the release
of cell therapy products can be a complex
process. Parameters such as viability, sterility,
and presence of endotoxin can be measured
using easily validated tests, whilst surface
phenotype is a more complex proposition.
In Birmingham, we have integrated a
MACSQuant® Flow Cytometer into our ATMP
manufacturing facility QC lab as a singleplatform cytometer for the enumeration
and phenotypic analysis of monocytes and
mature DCs.
Following apheresis, total leukocyte count
and percentage of monocytes are calculated
using the MC CD14 Monocyte Cocktail and
“Express mode” on the MACSQuant Analyzer
10 (fig. 1). This requires minimal operator
input and gives a rapid determination of
key parameters required for enrichment of
CD14+ monocytes using the CliniMACS
Platform. This same cocktail and protocol are
used after enrichment to determine absolute
monocyte count and purity before beginning
differentiation in culture.
Following differentiation of Mo-DC as
described previously, an analysis of phenotype
is made on day 6, prior to addition of antigen
and MPLA. Specifically, we measure the
Before separation
10³
Enriched CD14+ cells
10³
P1\P2\P3\P4
23.88%
10²
CD14-PE
CD14-PE
10²
CD14-PE
CD14-PE
P1\P2\P3\P4
99.09%
10¹
1
0
-1
-1 0 1
10¹
10²
CD45-VioBlue
CD45-VioBlue
10³
10¹
1
0
-1
-1 0 1
10¹
10²
10³
CD45-VioBlue
CD45-VioBlue
Figure 1 Enrichment of CD14+ monocytes. CD14+ monocytes were enriched from PBMCs using the
CliniMACS System. The percentage of monocytes was calculated using the MC CD14 Monocyte Cocktail and
“Express mode” on the MACSQuant Analyzer 10.
22
MACS & more
Vol 17 • 1/2016
expression of HLA-DR, CD11c, CD86 and
CD14 (fig. 2). Final release of the cells is
performed on day 8 with samples taken for
sterility testing, endotoxin contamination and
repeat phenotypic analysis. Our release criteria
stipulate an increase in expression of HLADR and CD86 on dual-positive cells between
day 6 and day 8, consistently high expression
of CD11c, and absence of CD14. Cells that
meet these criteria are released in four
batches. An initial batch will be delivered
directly to the patient for administration via
the hepatic artery whilst the remaining three
batches are resuspended in an appropriate
volume of cryoprotectant (CryoStor® CS10,
BioLife Solutions®) before being transferred to
individual CryoMACS® Freezing Bags (Miltenyi
Biotec). Controlled-rate freezing is carried out
according to a previously validated protocol
and cells are stored in a –152 °C mechanical
freezer until required. Repeat peripheral
infusions will be thawed at the patient bedside
and delivered via a peripheral vein monthly for
3 months.
Transfer to GMP and validation
We are currently completing the final phase
of validations to begin delivery of this Mo-DC
cancer vaccine in the near future. Engineering
runs under full GMP conditions are underway
using both the CliniMACS Plus Instrument
and the CliniMACS Prodigy®, though
intention is to use the CliniMACS Prodigy
for all cell enrichments. This automation of
the enrichment process is a key forward step
in the manufacturing of Mo-DC therapies,
saving considerable hands-on time and being
relatively simple to scale out with the provision
of additional machines. Furthermore, as the
development of the CliniMACS Prodigy
continues, the introduction of a compliant
culture process to take patient apheresis
samples through to immature Mo-DCs without
the need for additional manual manipulations
will simplify the delivery of novel cellular
therapies.
Conclusion
In common with many similar facilities
throughout the UK, our experience in
Birmingham of developing an ATMP
manufacturing facility has been challenging.
The commitment of a strong team has enabled
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PERSPECTIVES
This article represents independent research funded by the
National Institute for Health Research (NIHR). The views
expressed are those of the authors and not necessarily those of the
NHS, the NIHR, or the Department of Health.
Day 6 immature Mo-DCs
52.46%
10²
10¹
1
0 0.02%
-1
-1 0 1
10³
47.50%
CD11c-APC
CD11c-APC
Anti-HLA-DR-VioBlue
Anti-HLA-DR-VioBlue
10³
0.02%
10¹
10²
10³
95.58%
4.38%
10²
10¹
1
0
0.04%
-1
-1 0 1
CD86-PE-Vio 770
CD86-PE-Vio
770
0.00%
10¹
10²
10³
CD14-VioGreen
CD14-VioGreen
10²
10¹
1
0 0.03%
-1
-1 0 1
0.00%
10¹
10²
10³
CD86-PE-Vio 770
CD86-PE-Vio
770
With a rapidly increasing incidence of liver
disease in the UK, the development of novel
therapies has never been more pressing.
Moreover, as we gather a greater understanding
of the interplay between the human immune
system and tumour microenvironment we have
at our disposal a greater armoury to manipulate
the immune response to cancer. For many years
cell therapy has promised much, but failed to
deliver comprehensively. Combination of
improved manufacturing techniques with
immunomodulation is moving the field into
an exciting era that may finally see us translate
some of the early potential into meaningful
miltenyibiotec.com
CliniMACS Prodigy
200-075-301
CliniMACS CD14 MicroBeads
130-019-101
CliniMACS CD14 Reagent
272-01
autoMACS Pro Separator
130-092-545
Human GM-CSF –
premium grade (100 µg)
130-093-866
170-076-302
MACS GMP Cell Expansion Bags
170-076-403
gentleMACS Dissociator
130-093-235
gentleMACS M Tubes
130-096-335
10²
MACSQuant Analyzer 10
130-096-343
10¹
Anti-HLA-DR-VioBlue, CD11c-APC,
CD86-PE-Vio 770, CD14-VioGreen
*
CryoMACS Freezing Bags
**
93. 00%
6.87%
MC CD14 Monocyte Cocktail, human 130-092-859
1
0
0.13%
-1
-1 0 1
0.00%
10¹
10²
10³
CD14-VioGreen
CD14-VioGreen
Figure 2 Phenotypic characterization of mature Mo-DCs. Enriched CD14+ cells were differentiated for
6 days and the resulting immature Mo-DCs were matured for two days. Cells were stained with Anti-HLA-DRVioBlue®, CD86-PE-Vio® 770, CD11c-APC, and CD14-VioGreen™ on day 6 and day 8, and analyzed by flow
cytometry on the MACSQuant Analyzer 10.
us to bring the ImmunoTACE clinical trial into
reality and has demonstrated how research
laboratory tools can, with careful planning,
be integrated into validated manufacturing
processes for the preparation of ATMPs.
151-01
DendriMACS GMP Medium
10³
74.11%
CD11c-APC
CD11c-APC
Anti-HLA-DR-VioBlue
Anti-HLA-DR-VioBlue
25.86%
Order no.
CliniMACS Plus Instrument
Human IL-4 – premium grade (100 µg) 130-093-922
Day 8 mature Mo-DCs
10³
MACS Product
treatments for the many patients living with
the burden of cancer.
References
1. Llovet, J. M. et al. (2002) Lancet 359: 1734–1739.
2. Lo, C. M. et al. (2002) Hepatology 35:
1164–1171.
3. Chen, C. H. et al. (2001) Cancer Lett. 164:
189–195.
4. Sideras, K. et al. (2015) Br. J. Cancer 112:
1911–1920.
5. Yin, Y. H. et al. (2005) Br. J. Cancer 93: 458–463.
6. Palmer, D. H. et al. (2009) Hepatology 49:
124–132.
7. Greten, T. F. et al. (2010) J. Immunother. 33:
211–218.
8. Hato, T. et al. (2014) Hepatology 60: 1776–1782.
9. Zitvogel, L. and Kroemer, G. (2012)
Oncoimmunology 1: 1223–1225.
* Visit www.miltenyibiotec.com/antibodies
** Visit www.miltenyibiotec.com/cryomacs
The autoMACS Pro Separator, gentleMACS Dissociator
and gentleMACS Tubes, MACS Premium-grade Cytokines,
MACSQuant Analyzer 10, MC CD14 Monocyte Cocktail, AntiHLA-DR-VioBlue, CD11c-APC, CD86-PE-Vio 770, and CD14VioGreen are for research use only.
The CliniMACS® System components, including Reagents, Tubing
Sets, Instruments, and PBS/EDTA Buffer, are manufactured
and controlled under an ISO 13485–certified quality system.
In the EU, the CliniMACS System components are available as
CE-marked medical devices. In the US, the CliniMACS CD34
Reagent System, including the CliniMACS Plus Instrument,
CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and
LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved;
all other products of the CliniMACS Product Line are available
for use only under an approved Investigational New Drug
(IND) application or Investigational Device Exemption (IDE).
CliniMACS MicroBeads are for research use only and not for
human therapeutic or diagnostic use.
MACS® GMP Products are for research use and ex vivo cell
culture processing only, and are not intended for human in vivo
applications. For regulatory status in the USA, please contact your
local representative. MACS GMP Products are manufactured and
tested under a quality management system (ISO 13485) and are
in compliance with relevant GMP guidelines. They are designed
following the recommendations of USP <1043> on ancillary
materials. No animal- or human-derived materials were used for
manufacture of these products.
CryoMACS® Freezing Bags are manufactured by Miltenyi Biotec
GmbH and controlled under an ISO13485 certified quality
system. These products are available in Europe as CE-marked
medical devices and are marketed in the USA under FDA 510(k)
clearance.
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
Acknowledgments
We acknowledge the NIHR for their funding support of this work
and the team in the Advanced Therapies Facility at the University
of Birmingham, in particular Heather Beard and Savita Mehmi for
their assistance in validation of release tests.
Vol 17 • 1/2016
MACS & more
23
The MACSQuant® Tyto™
The revolution in cell sorting has begun
The MACSQuant® Tyto™ is revolutionizing
cell sorting. Our patented microchip-based
technology opens new possibilities in basic
research and clinical settings with high-speed
multiparameter flow sorting in the safety of a
fully enclosed cartridge.
Innovation
Sort cells with the world’s fastest mechanical sort
valve and 11-parameter fluorescence-based sorting.
Safety
Samples and operator are kept contamination-free
and safe with disposable, fully enclosed cartridges.
Viability
Cells are gently driven through the microchip with
low positive pressure. Less stress means higher yield
of viable, functional cells.
miltenyibiotec.com/tyto
The MACSQuant Tyto is for research use only.
Ease of use
No droplet delay or laser alignment needed. Simply
insert the cartridge, gate on cells, and sort.
PERSPECTIVES
Engineering human cells with lentiviral vectors:
Making an impact on human disease
Rimas J. Orentas
Translational Research and Development, Lentigen Technology Inc., A Miltenyi Biotec Company
Tackling disease with engineered
immune cells
Advances in science are also advances in
human self-understanding. When tragedy such
as life-threatening infection or advanced cancer
strikes, we look both without, to pharmacologic
interventions, and within. Within our own
bodies there is a network of systems that govern
tissue development and repair, inflammation,
and the response to microorganisms.
Depending on our own scientific training
and the purpose for which we intervene, we
approach these largely unknown systems with a
particular bias, and often with a desire to meet
a pressing medical need. Miltenyi Biotec has
been enabling investigators in biomedicine to
unravel these mysteries by providing key tissue
preparation and cell separation reagents. With
these tools investigators can isolate and study
stem cell populations, the cell populations that
govern inflammation and wound repair, and
the various cells that play a role in the immune
response to both infectious agents and cancer.
Vaccination against viral and bacterial
pathogens has been a pillar of public health
for decades. However, vaccination works best
in disease prevention, as opposed to a direct
therapeutic intervention. Standard vaccine
approaches to chronic viral infections such as
HIV or hepatitis C have not been effective to
date. And the same can be said for cancer. In
light of this, scientists are working to unravel
the mechanisms and principles that define the
immune response to vaccines, and have begun
to apply them to treating established human
miltenyibiotec.com
Lentiviral genome
Tev
Rev
LTR
Gag
Tat
Vif
Pol
Vpr
LTR
Vpu
Env
Nef
Split packaging & SIN
SIN
LTR
PGKp
CAR
SIN
LTR
+
Gag
Pol
Rev
+
Env
Split packaging & SIN & 4-plasmid system
SIN
LTR
PGKp
CAR
SIN
LTR
+
Gag
Pol
+
Rev
+
Env
Figure 1 Evolution of human lentiviral gene vectors. The identification of HIV was a medical breakthrough
that allowed significant advances in treatment to be made. This discovery also introduced us to a powerful tool
for introducing synthetic DNA sequences into the human genome. On the top row, the critical genes expressed
by the wild-type virus are shown. This virus was transformed (or "gutted") into a non-infectious vector that
cannot replicate through a number of steps. First, in order to assemble a lentiviral vector particle (LV) a
producer cell is transfected with plasmid DNA that contains the minimal information required to assemble a
particle that can bud from the cell and transduce a target cell, without ever being able to reactivate. In the middle
row the classic three-plasmid system is depicted. Here the LTR that normally drives gene expression in HIV has
been modified such that once it incorporates into the genome it is “self-inactivating” (i.e. a SIN LTR) and no
longer functional. This requires a second synthetic promoter, shown here is one from the housekeeping gene
PGK, to drive the therapeutic gene of interest. This is the only part of the vector that inserts into the genome.
On a second plasmid the gag (group antigens, the virion structural proteins), pol (polymerase, i.e. reverse
transcriptase), and the rev element are encoded. The third plasmid encodes env (envelope), which for most
LV is derived from vesicular stomatitis virus (VSV-G). An added level of safety was created in the 4-plasmid
system, bottom row, where the rev element was removed and is expressed from its own plasmid. To re-create
an infectious agent all of these elements would need to re-combine correctly, the SIN promoter would have to
be repaired, and a new env provided, which is an essentially impossible event.
cancers. It is abundantly clear that vaccination
induces and expands activated lymphocyte
populations in the body, and that the transfer of
vaccine-educated T lymphocytes can transfer
protective immunity. In some approaches,
native T cell populations are used. The primary
example of this would be the expansion ex
vivo and subsequent re-infusion of tumor-
Vol 17 • 1/2016
MACS & more
25
PERSPECTIVES
infiltrating lymphocytes, recently reviewed by
Rosenberg and Restifo¹. In other approaches,
immune cells are first genetically modified.
The ability to genetically modify, or engineer,
immune cell populations is central focus for
Lentigen Technology, Inc., a Miltenyi Biotec
Company. Lentigen produces lentivirus-based
gene vectors. These gene vectors are essentially
highly modified versions of the HIV virus.
Because the modified virus cannot replicate, or
make new copies of itself, the term "vector" as
opposed to virus or virion is used. This is an
important distinction, as it makes clear that we
have a very safe and controlled way to introduce
new DNA sequences into the human genome,
see figure 1. The DNA being introduced into
the genome can encode for a native human
transcript, such as the alpha and beta chains
of the T cell receptor (TCR) for antigen, or
can encode for new types of proteins that are
a composite of different human transcripts
and thus are termed "synthetic" or "chimeric".
Although not encoded in toto by the human
genome, these are still essentially self-proteins,
created from distinct functional protein
subdomains. If the individual components
were not encoded by the genome, the protein
itself would be foreign, and thereby a locus
for immune rejection of the cell that expresses
it. This would then eliminate any potential
therapeutic benefit of the modified cell
population.
Engineering of T cells with chimeric
antigen receptors
Contemporary thinking about synthetic
biology, in fact the very use of the term,
was crystallized by Wacław Szybalski² who
envisioned a new biology wherein genetic
control circuits could be engineered at the
level of the cell and subsequently the entire
organism. With regard to proteins associated
with immunologic function, Zelig Eschar
demonstrated that receptor- or ligandbinding domains, transmembrane sequences,
and intracellular signaling motifs could be
interchanged between different cell surface
glycoproteins³. These patchwork proteins were
soon termed "chimeric antigen receptors" or
CARs. Putting these advances together, we have
now arrived at the current era where control
of gene expression with molecularly cloned
promoter and enhancer elements, and the
creation of novel chimeric proteins (fig. 2), can
26
MACS & more
Vol 17 • 1/2016
Synthetic biology CAR shop
CAR elements
A) Active CARs
B) Double CARs
C) Subunit CARs
D) iCARs
αCD22 scFv
αCD19 scFv
αCD20 scFv
Sp6 scFv
CD8 linker (w/TM)
2nd gen. CAR, CD137/CD3ζ
CD28 TM, signaling
Just CD3ζ
Just costim.
domains
E) CID assembled “AND” CARs
(GGGGS)x
T cell inhibitory domain
dimerization agent
Figure 2 The chimeric antigen receptor. The expression of a chimeric antigen receptor (CAR) on the surface
of a LV-transduced T cell requires a binding domain, scFv, to interact with the tumor-expressed surface antigen
on the opposing cell membrane, and a means to link that binding to T cell–specific transmembrane and
signaling domains. Here we highlight a subset of the tools that can be assembled for the engineering of a T cell
with CAR. Shown on the left are i) different scFv-derived binding domains (anti-CD19, 20, 22, Sp6 control), ii)
a linker that has been used to join these domains to T cell signaling domains (CD8 linker with transmembrane
domain, TM), iii) two signaling systems that have been used to activate T cells (the second-generation CAR that
incorporates the activation domains present in the CD137 and CD3ζ chain molecules or a domain that includes
the CD28 transmembrane and activation domains), iv) an amino acid linker sequence (GGGGS) composed
of a string of glycines and serines that is multimerized up to 4 times to join individual protein domains, v) a
representative inhibitory domain that could be derived from PD-1 or an intracellular phosphatase and vi) a
representative chemical dimerization reagent. These subunits can be assembled as A) active CARs, that can lyse
tumor cells upon contact, B) double CARs that contain two binding domains, C) subunit CARs that require
multiple contacts to give full T cell activation, D) iCARs, inhibitory CARs that turn off T cells upon binding
to a target antigen, and E) "AND" CARs that require assembly with a chemically induced dimerization (CID)
agent. These next-generation, gated "AND" CARs require both the target and the CID to be present for activity.
be introduced together as a unit into human
cells in a safe manner and with apparent
clinical benefit, (reviewed in ref. 4). Thus the
engineering of T cells with CARs is the result
of many independent contributions in science
that span protein chemistry, cell biology, gene
vector biology, and adoptive immunotherapy.
In looking to the future, what are the key areas in
which synthetic biology will be used to improve
immunotherapeutic cell populations? The first
will be in the cellular substrate itself. The second
will be in better defining target antigens of
interest. The third will be in better design of the
effector molecule expressed by the engineered
cell. The final area we will consider will be the
manner in which the transgene and the cell
expressing that transgene are controlled (fig. 3).
miltenyibiotec.com
PERSPECTIVES
Initial cell type
Engineered T cell
Gene vector
TSCM
Next-generation
“gated” CARs
TCM
X
Selected CD4+
and/or CD8+ T cells
Transgene
expression
CARs
Virus-specific T cells
Soluble factors
Target selection
X
Tumor vs. normal
•
•
•
•
Genetic control elements
CID activation or cell death
Expression profiling
Next-generation sequencing
Proteomic analysis
Tumor-specific Abs
Figure 3 The engineered T cell. To create an engineered cell we begin with a target cell population that we
wish to modify, and a specific antigen that can be targeted. At the top left, several cell types are illustrated
from which CAR strategies have been approached (T stem cell memory (Tscm), T central memory (Tcm),
T cells that have been selected by surface marker expression, and T cells that are specific for viral antigens,
and can be expanded in vitro). At the bottom left, we illustrate the concept that understanding what serves as
a suitable target is an essential aspect of CAR therapy. The more restricted to the cancer cell, and the greater
the over-expression of a tumor-associated protein, the higher the margin of safety for CAR-based therapy. The
engineering of the T cell itself begins with the introduction of a gene vector (such as a LV) that encodes the antitumor effector protein (CAR), as well as other factors that control the biology of the T cell itself. These factors
include soluble proteins that enhance T cells or alter the tumor microenvironment, components of a genetic
switch, and domains that can induce cell death as a means to provide a safety switch. Though not illustrated,
T cell costimulatory domains or agents that block immune checkpoints can also be envisioned.
As currently applied, adoptive immunotherapy
with engineered T cells is usually performed
using unselected lymphocyte populations.
However even in these procedures, there is
some degree of enrichment for activated T cells.
For example, in the creation of CD19-specific
CAR T cells for the treatment of pediatric
B-ALL, the lymphocyte population (obtained
by apheresis) is stimulated with magnetic
beads decorated with anti-CD3 and anti-CD28
antibody⁵. During activation and culture,
lymphocytes bound to these beads are enriched
by adherence to a magnet. In an attempt to
standardize this process and improve the T cell
populations used for CAR T cell engineering,
Sommermeyer et al., described the most active
CD4+ and CD8+ T lymphocyte populations
miltenyibiotec.com
that could be selected and subsequently
infused for a consistent therapeutic effect⁶.
Thus, immunomagnetic bead selection plays
a key role in identifying the appropriate T cell
population for adoptive transfer. Investigators
are also looking to include more naive T cell
subsets that appear to be active at lower
numbers and which expand and persist to a
greater degree once infused into the host⁷.
The cells used for adoptive immunotherapy
can be engineered to express costimulatory
molecules such as CD137L, cytokines, or
cytokine receptors, and also microRNA species
associated with increased T cell survival and
anti-tumor reactivity⁸,⁹. The ability to express
multiple transcripts from a lentiviral vector
(LV) makes the ability to engineer T cell
populations a single-step process. Furthermore,
although the ability to augment anti-tumor
T cell populations in vivo with antibody-based
approaches, such as anti-PD-1 or anti-CTLA-4,
has been quite effective, similar effects can be
seen through the knockdown of the appropriate
receptor on the engineered T cell population¹⁰.
Thus, LV-based T cell engineering can not only
deliver a payload into the genome, such as a
CAR, but also transcripts that will govern the
activation, expansion, and persistence of the
therapeutic cell population.
Identifying appropriate CAR T cell targets
How we search for a truly tumor-specific
antigen (TAA, tumor-associated antigen),
has changed due to the impact of CAR T cell
therapy. Traditionally a TAA is a mutated-self
or a foreign viral protein that is expressed by
a cancer cell. To date, the most effective CAR
target, CD19, is a normal self-protein that is
overexpressed on the leukemia cell surface.
When CAR-modified T cells that recognize
CD19 are infused, the normal B cell population
that expresses this antigen is also eliminated.
This is an "on-target" but "off-tumor" effect.
The loss of normal B cells is a survivable
event that can be managed with polyclonal
immunoglobulin infusions. This may not be
the case for other self-antigens, and they will
have to be considered on a case-by-case basis.
Until this year, the antigen-binding domain
of the CAR, the scFv domain, was often not
the target of affinity maturation, as is the case
for antibody-based therapeutics. Studies by
Chmielewski et al. demonstrated that upon
reaching a threshold affinity, not much is gained
in affinity maturation of the antigen-binding
domain of the CAR, as the T cell transduced
to expressing the CAR expresses numerous
copies of the protein on the plasma membrane,
and avidity thus trumps affinity¹¹. However,
a very important recent study showed that
degrading the scFv affinity to the level where
the avidity drives the biology of the CARexpressing T cell, may allow effective targeting
of tumor cells that over-express the TAA and
spare normal cells that express far fewer copies.
This was demonstrated using CARs based on
two antibodies specific for EGFR, low-affinity
nimotuzumab and high-affinity cetuximab¹².
This has invigorated genomic approaches to
searching for TAA based on comparing the
expression of normal to cancer-associated
Vol 17 • 1/2016
MACS & more
27
PERSPECTIVES
tissue samples by gene expression profiling or
next-generation sequencing methodologies¹³.
Switching CAR T cells on and off
If the approach to a specific cancer target
is still limited by the lack of a safe overexpressed target, this calls for a more complex
CAR structure to be developed that requires
two specific "hits" for activation, or which is
negatively regulated by an antigen that is overexpressed on a vital normal tissue. Neither of
these options has been put into practice in the
clinic, but experimental results are promising.
For negative signals both intracellular
phosphatases such as CD45 and the
intracellular signaling motifs of the checkpoint
molecules PD-1 and CTLA-4 have been
proposed¹⁴,¹⁵. Thus a CAR specific for TAA
"A", to which an active intracellular domain is
spliced, will react to any cell that expresses "A",
unless antigen "B" is present, which is targeted
by a second CAR expressed by the same T cell,
yet to which negative or inhibitory signaling
domains are spliced. There also have been
numerous "split CAR" approaches, wherein
binding to "A" delivers only a partial signal, and
full activation to the point of target cell cytolysis
requires a second binding event, "C", that is
also present on the tumor cell. This process
can also be approached pharmacologically,
wherein a CAR specific for "A" does not
contain any T cell activation signals, but does
contain a small-molecule binding domain that
can induce protein-protein association. In the
same LV a second transcript, "D", is expressed
that would contain the dimerization domain
linked to the intracellular signaling motifs that
would activate a T cell. This small molecule
thus becomes an "on switch" and is required
for full CAR activity. Chemical inducers of
dimerization (CID domains) have been widely
developed using the rapamycin/FK506 and
FK506-binding protein (FKBP) system. Similar
systems have been recently reviewed by Wu
et al.¹⁶.
The control of CAR T cells to date has employed
various mechanisms to immuno-deplete the
cells from the body, or to induce apoptotic
cell death. Immunodepletion is envisioned by
the use of an antibody that has already been
approved for therapy and has an established
safety profile. Creating a LV that expresses both
a CAR of interest and the extracellular domain
28
MACS & more
Vol 17 • 1/2016
of CD34, CD20, EGFR, or LNGFR allows for
a planned removal of the CAR-transduced
cell, should unwanted side-effects develop, by
infusion of antibody specific for these domains.
The rapidity by which this would occur in
the clinic is not yet known, and no reports
of clinical depletion of CAR T cells with an
antibody have been published. Nevertheless, it
may provide some degree of assurance in the
case of a risky target. The most effective "off
switch" to date is the induction of apoptosis by
a CID. In CAR-expressing T cells engineered
to express an engineered caspase 9 gene, the
small molecule AP109 induces dimerization
and rapidly induces cell death through the
activation of the endogenous apoptotic cell
death pathway, known as the iCasp9 system¹⁷.
A molecular "on switch" as envisioned by early
synthetic biologists, may require added levels of
control to prevent "read-through" transcription.
A eukaryotic version of a true genetic switch
mechanism, inducible by a small ligand, has
already been reported¹⁸. The challenge remains
to fully operationalize this system using
native human elements and clinically relevant
inducer molecules. To date these have all been
hormone-like small molecules that can interact
with distinct intracellular receptor domains
linked to transcriptional transactivators.
A bright future for synthetic biology
In conclusion, the era of synthetic biology
is upon us. The human T cell can be readily
engineered with lentiviral gene vectors to
express effector proteins and RNA molecules
ex vivo, and upon expansion these cells can
be re-infused into the body for therapeutic
effect. There have been promising reports of
children and adults with refractory B-ALL who
survived because engineered T cells were made
available for their treatment.¹⁹,²⁰ Our vision
is to expand this availability to any setting
where cell processing can be carried out, and
to continuously improve the targets identified
for CAR therapy. We hope to support and
participate in new technological developments
that allow investigators to control the function
of CAR proteins at the molecular level and to
control the expansion or the elimination of
T cells at the cellular level.
References
1. Rosenberg, S.A. and Restifo, N.P. (2015) Science
348: 62–68.
2. Szybalski W. and Skalka, A. (1978) Gene 4:
181–182.
3. Stancovski, I. et al. (1993) J. Immunol. 151:
6577–6582.
4. Lee, D. et al. (2012) Clin. Cancer Res. 18:
2780–2790.
5. Lee, D. et al. (2015) Lancet 385: 517–528.
6. Sommermeyer, D. et al. (2015) Leukemia Sep 15:
Epub ahead of print. doi: 10.1038/leu.2015.247.
7. Gattinoni, L. et al. (2012) Nat. Rev. Cancer 12:
671–684.
8. Pegram, H. et al. (2012) Blood 119: 4133–4141.
9. Ji, Y. et al. (2015) Proc. Natl. Acad. Sci. USA 112:
476–481.
10.Condomines, M. et al. (2015) PLoS One 10:
e0130518. doi: 10.1371/journal.pone.0130518.
11.Chmielewski, M. et al. (2011) Gene Ther. 18:
62–72.
12.Caruso, H. et al. (2015) Cancer Res. 75:
3505–3518.
13.Orentas, R. et al. (2014) Front. Oncol. 4: 134. doi:
10.3389/fonc.2014.00134.
14.James, J.R. and Vale, R.D. (2012) Nature: 487:
64–69.
15.Federov, V. et al. (2013) Sci. Transl. Med. 5:
215ra172. doi: 10.1126/scitranslmed.3006597.
16.Wu, C. et al. (2015) Science 350: aab4077. doi:
10.1126/science.aab4077.
17.Straathof, K. et al. (2005) Blood 105: 4247–4254.
18.Deans, T. et al. (2007) Cell 130: 363–372.
19.Grupp, S.A. et al. (2013) N. Engl. J. Med. 368:
1509–1518.
20.Maude, S.L. et al. (2014) N. Engl. J. Med. 371:
1507–1517.
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130-110-132
The illustration shows a natural killer (NK) cell attacking
a tumor cell. NK cells are an important part of the innate
immune system, playing a key role in host defense
against cancer, for example.