Impact Objectives• Investigate how inhibiting T cell exhaustion can improve immunotherapy• Research on energy metabolism and epigenetic control in T cells

A protocol forT cell immunotherapy

Treating cancer via
T cells immunotherapy
At Ehime Prefectural University of Health Sciences, researchers are exploring a revolutionary new way totreat cancer that involves suppressing T cell exhaustion during T cell immunotherapy

By exploring and better understanding the process of T cell exhaustion, Professor Takeshi Yamada isseeking to develop treatments for refractory cancerP

How did you
become interestedin this area ofresearch?
I began studying
chronic infectious diseases, such as HIV andhepatitis C virus infections, as a virologist at
the University of Tokyo in 1996. During thattime, I learned that T cells in these patientsare exhausted due to sustained antigen
stimulation and other factors, which leads
to T cell dysfunction against viruses. This
also led me to become interested in T cell
exhaustion which is problematic in refractory
cancer. My turning point was when I decided
to study abroad in the US. This was when
I started my career as an immunologiststudying CD8 T cell differentiation at Baylor
College of Medicine in 2005. Since then,I have been researching exhaustion anddifferentiation of CD8 T cells at Ehime
University (2013 to 2018) and now at Ehime
Prefectural University of Health Sciences(since 2018).
What are some of the big gaps in ourknowledge about the function andmechanism of the immune system that you
are hoping to fill with your studies?
We are working to elucidate how T cells
become dysfunctional in order to improve T
cell immunotherapy by developing a method
to inhibit exhaustion. In order to do this,06 www.impact.pub

we have been studying the mechanisms ofT cell differentiation related to exhaustion
through focusing on energy metabolism andepigenetic control in T cells.
Immunotherapy is a very innovative way
to treat patients with refractory cancer.
However, its effect is limited in somepatients, partly because T cell exhaustion isinduced with high expression of inhibitory
receptors (known as immune checkpoint
molecules) such as PD-1, LAG3 andCTLA-4. We need to know the reasons whyexhaustion occurs in patients in order toimprove T cell immunotherapy. This is themain focus of our efforts in our research.
From your perspective, what makes
your research novel in regards tosuppressing T cell exhaustion during T cell
immunotherapy?
We are using innovative methods that willraise the versatility of protocols against
many kinds of cancers. It is a new idea tosuppress T cell exhaustion and improve thequality of T cells by culturing with drugs. Our
method is quite simple as it involves adding
drugs that have already been used into acell culture to control intracellular energy
metabolism or epigenetic enzymes.
We are developing new protocols tosuppress exhaustion using comparatively
convenient ways such as drugs that can beadded into T cell culture before adoptivelytransferring them to cancer patients. Our
methods will be applicable to many typesof cancer as long as the cause of failure ofimmunotherapy is due to the exhaustion ofT cells. So, in the first instance, patients withrefractory cancer will likely benefit from ourprotocols.
Can you talk a little about some of theresults you have seen so far with yourexperiments?
Our preparative experiments have shown
some positive results, including thattumour-specific T cells cultured with drugs
to inhibit glutamine metabolism decreased
the tumour growth and increased thesurvival of mice carrying tumour cells,
compared to the control without drugs.
These results indicate promising outcomesof the new protocols we are developing.
How are you hoping to build on what you
have discovered through your research sofar?
Our next steps will be to find the best
conditions to culture tumour-specific T
cells using drugs to control intracellularenergy metabolism or epigenetic change inT cells in order to gain the largest effects on
anti-cancer therapy. Next, we will focus on
applying our methods from mouse model tothe human body. lrofessor Takeshi Yamada has dedicatedhis career to this field. He is based at
the Department of Medical Technology,
Immunology, Ehime Prefectural Universityof Health Sciences in Japan where he isleading a team seeking to elucidate themechanisms of T cell exhaustion in order toimprove T cell immunotherapy. He explainsthat adoptive immunotherapy, in which Tcells are taken from a patient and grownin a laboratory in order to increase thenumber of tumour-specific T cells before
being returned to the patient, can be used
to treat intractable cancers by increasing thenumber of T cells on hand to fight the cancer,
thereby boosting the immune system’s
cancer-fighting ability. One barrier that
researchers are encountering, though, is thatonce outside of the body, the T cells can fall
into a state of exhaustion and are therefore
unable to provide effective treatment. As
the mechanisms of T cell exhaustion aren’t
well-known, this is a hurdle that needs to beovercome.
Yamada and his team want to find a way toinhibit T cell exhaustion, and their studies arefocusing on intracellular energy metabolism
and epigenetic control (epigeneticmechanisms regulate biological processes)
in T cells using mouse models. ‘We aredeveloping protocols to improve T cellfunction for immunotherapy by controlling
epigenetic changes involved in glutamine
metabolism, which induces T cell exhaustion,’
he outlines. Ultimately, they want to improvethe culture method of T cells in adoptive
immunotherapy in order to suppress Tcell exhaustion. ‘This is a novel approach,with previous research having focused on
activating and proliferating tumour-specific
T cells,’ he says. In addition, the team’s focus
on epigenetic control is an innovative angle.
Overall, this novel work has the potential to
make significant inroads in the treatment ofcancers, particularly intractable cancers.
EPIGENETIC CHANGES AND CONTROLThe researchers are interested in the processof T cell differentiation which, simply put,is when a T cell transitions from one cell
type to another. They believe that T cell
exhaustion occurs after excess proliferationand differentiation when there are too many
nutrients and too much stimulation ofantigens or cytokines in the T cell culture.

‘The exhaustion must be linked with T celldifferentiation and senescence by epigenetic
changes via glutamine metabolites such as
α-ketoglutarate,’ Yamada explains.
As such, the researchers’ work surrounds
the mechanism of T cell differentiation
via intracellular energy metabolism andepigenetic changes and how this can impact
on exhaustion. ‘In previous research, theteam clarified that the enhancement ofglutamine metabolism that occurs during theactivation of T cell cultures causes epigenetic
changes that induce T cell exhaustion,’states Yamada. ‘Essentially, the glutamine
metabolite α-ketoglutarate acid triggers theactivation of a protein called histone H3K27
demethylase, which induces T cell exhaustion
through epigenetic changes,’ he confirms.
Yamada explains the science behind thisCampus photo of Ehime Prefectural University of Health Sciences
www.impact.pub 07

and the path his research is following. ‘We
have reported that exhaustion is caused
partially by epigenetic changes, such asactivation of histone H3K27 demethylase
using α-ketoglutarate as a cofactor α-ketoglutarate is a product of glutamine
metabolism,’ he explains. ‘We also reported
that the inhibitor of glutamine metabolic
enzymes or histone H3K27 demethylase
reduced exhaustion of cultured T cells.’ The
team will apply these studies to develop
new methods in order to inhibit exhaustion
and improve T cell immunotherapy using
drugs which restrict the activity of glutamine
metabolism or histone H3K27 demethylase.
‘Indeed, in our preliminary studies, the teamfound that adding the inhibitor of glutamine
metabolism or histone H3K27demethylase to

H3K27demethylase, and anti-tumour analysis
will be performed using a cancer mousemodel, with the tumour size measured overtime in order to verify the anti-tumour activityof the T cells. This way, the team will be able
to decipher the optimum culture conditions.
‘As part of experiment two, we will then collect
the transferred T cells infiltrating the tumour
from the mice and perform an analysis toclarify the mechanism of enhancing antitumour activity by adding drugs to the culture,’
explains Yamada. The team also intends toperform culture experiments using T cells
from humans in order to see if the same
results are observed. ‘In experiment three,
in order to clarify the effects of combined
treatment, a kinase inhibitor or monoclonal
antibody drug will be administered to tumourbearing mice that have undergone adoptive

We are developing protocols to improve T cell function
for immunotherapy by controlling epigenetic changes
involved in glutamine metabolism, which induces T cell
exhaustion
CD8 T cell cultures enhances differentiation
into unexhausted long-lived T-cell as known
as immunological memory,’ says Yamada.
They are now expanding on this finding inorder to develop a method to suppress T
cell exhaustion via epigenetic control. This
work covers three experiments involving CD8T cells, otherwise known as ‘killer cells’ fortheir tumour killing ability, conducted overa three-year period. ‘The first of the threeexperiments is an exploration of the optimal
culture conditions for enhancing anti-tumour
activity via epigenetic control,’ highlightsYamada. ‘In the second experiment, theresearchers will perform a detailed analysis
on cell proliferation and differentiation using
cultured cells and will clarify the mechanism
behind enhancing anti-tumour activity,’ hesays. Finally, in the third experiment, Yamada
and the team will look into how combination
treatments with molecular drugs, that arecurrently used to treat cancer, can be used tofurther enhance the effect of epigenetic control
on anti-tumour activity.

immunotherapy. Finally, the researchers willpropose a new immunotherapy protocol using
an epigenetically controlled culture containing
histone H3K27 demethylase inhibitor,’ heconcludes.

ANTI-TUMOUR ACTIVITY
In experiment one, the researchers will
culture tumour-specific CD8 T cells using theinhibitor of glutamine metabolism or histone

A LIFE-SAVING PROTOCOLThe next steps for the researchers will beto translate their mouse model findings tohumans. ‘We will confirm the effects of drugs

08 www.impact.pub

This research involves the important
contributions of valuable collaborators, withexpertise in mouse models, translational
research and T cell differentiation. ‘I amcollaborating with Dr Yuya Arakwa, who is
an assistant professor in our team and a key
person on our project. He plays a critical rolein experiments and analyses using mousemodels,’ comments Yamada. The team arealso collaborating with Dr Masaki Yasukawa
and Yoshiaki Norimatsu at Ehime Prefectural
University of Health Sciences on translational
research for cancer patients, Dr Masakatsu
Yamashita at Ehime University for important
discussions on T cell differentiation, and Dr
Yuki Imai in Ehime University and Dr Hirotake
Tsukamoto in Kyoto University on the workwith mouse models.

using human T cells in the culture and thenconfirm the effects and safety of our methodin the human body by injecting cultured cells,’
says Yamada. ‘We believe that our method
is safe as it doesn’t involve directly injecting
drugs into the human body.’ If the researchers
are successful in proposing a protocol for T
cell immunotherapy with higher anti-tumour
effect than has been seen previously, this
could lead to an effective treatment for many
cancers, including those that are intractable.
Ultimately, this could revolutionise cancer
treatment and save lives. lProject Insights
FUNDINGJSPS KAKENHI, Research Grant of thePrincess Takamatsu Cancer Research Fund,Takeda Science Foundation andITO EN, LTD.
COLLABORATORS
Ehime Prefectural University of Health
Sciences –Professor Masaki Yasukawa andProfessor Yoshiaki Norimatsu
Ehime University - Professor Masakatsu
Yamashita and Professor Yuki Imai
Kyoto University - Program-Specific Associate
Professor Hirotake Tsukamoto
TEAM MEMBERS
Ehime Prefectural University of Health
Sciences – Assistant Professor Yuya
Arakwa and Graduate student Yuzuki
Tano
CONTACT DETAILS
Professor Takeshi Yamada
T: +81 89 958 2111
F: +81 89 958 2177
E: tyamada@epu.ac.jp
W: https://www.epu.ac.jp/index.html
BIO
Professor Takeshi Yamada has worked at
the University of Tokyo, Baylor College ofMedicine and Ehime University. Yamadacurrently works at the Department ofMedical Technology, Laboratory Technology,Immunology, Ehime Prefectural University
of Health Sciences. He is a member of theJapanese Society for Immunology and The
Japanese Cancer Association.