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Irradiated CD19 chimeric antigen receptor YTS cells retain
antitumor activity and offer a scalable alternative to
autologous CAR-T therapy
by Hanan Jaber, Anas Abu khalaf, Lola Weiss, Itay Cohen, Natali Bannoura, Tamara Osaili, Ortal Maximov,
Abigael Eva Chaouat, Mark Kournos, Adi Tabib, Dror Mevorach, Yarden Engel, Michael Berger,
Ofer Mandelboim and Shlomo Elias
Received: November 4, 2025.
Accepted: July 27, 2026.
Citation: Hanan Jaber, Anas Abu khalaf, Lola Weiss, Itay Cohen, Natali Bannoura, Tamara Osaili,
Ortal Maximov, Abigael Eva Chaouat, Mark Kournos, Adi Tabib, Dror Mevorach, Yarden Engel,
Michael Berger, Ofer Mandelboim and Shlomo Elias. Irradiated CD19 chimeric antigen receptor YTS cells
retain antitumor activity and offer a scalable alternative to autologous CAR-T therapy.
Haematologica. 2026 Aug 13. doi: 10.3324/haematol.2025.300165 [Epub ahead of print]
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1
Irradiated CD19 chimeric antigen receptor YTS cells retain antitumor activity and
offer a scalable alternative to autologous CAR-T therapy
Hanan Jaber1,2*, Anas Abu khalaf1,2*, Lola Weiss1, Itay Cohen1,2, Natali Bannoura1,2, Tamara
Osaili,1,2 Ortal Maximov,1,2 Abigael Eva Chaouat 1,2, Mark Kournos2, Adi Tabib3, Dror
Mevorach3, Yarden Engel2, Michael Berger2, Ofer Mandelboim2 and Shlomo Elias1,2
1 Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah
Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel
2 The Lautenberg Center for General and Tumor Immunology, Department of Immunology
and Cancer Research, Institute for Medical Research Israel Canada (IMRIC), Hebrew
University-Hadassah Medical School, Jerusalem Israel.
3 Institute of Rheumatology-Immunology-Allergology and Department of Medicine,
Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem,
Israel
* These authors contributed equally
Running title: Engineered YTS cells as a cellular therapy
Keywords: YTS, NK, CAR-CD19, immunotherapy
Details of corresponding authors: Correspondence should be sent to Shlomo Elias
selias@hadassah.org.il, Tel: 972-2-6757515, Fax: 972-2-6757227
Competing interests: The authors declare that they have no competing interests.
2
Author Contributions
HJ performed most of the in vitro experiments. AAK performed the mice experiments and
other experiments. LW assisted with mouse experiments. IC, NB, A EC, OM (Maximov) and
AT performed or assisted to experiments. MK generated the 2B4 knockout cells. AT and DM
assisted with dendritic cell experiments. YE and MB provided and assisted with melanoma cell
lines and related analyses. OM (Mandelboim) and SE conceptualized the study, SE supervised
the research. HJ and SE wrote and edited the manuscript. All authors provided critical
feedback, and approved the final version of the manuscript.
Data Sharing Statement
All data generated or analyzed during this study are included in this published article and its
supplementary information files. Any remaining raw data or protocols are available from the
corresponding author upon reasonable request.
Conflict of interest statement
H.J., A.A.K., O.M., and S.E. are listed as inventors on a patent application related to the use of
engineered YTS cells for immunotherapy (PCT Patent Application No. PCT/IL2024/050118).
The remaining authors declare no competing financial interests.
Acknowledgments
We thank Shlomit Kfir -Erenfeld and Natalie Asherie from the Department of Bone Marrow
transplantation at Hadassah Medical Center for assistance in the CAR-T production. We thank
Dana G. Wolf and Esther Oiknine -Djian from the Clinical Virology Unit, Department of
Clinical Microbiology and Infectious Diseases, for their assistance with the EBV experiments.
3
We thank Zhanna Yekhtin for assistance with the mice experiments. ChatGPT (OpenAI, San
Francisco, CA, USA) was used to improve the language and readability of the manuscript. The
authors reviewed and edited the content as needed and take full responsibility for the final text.
Funding
SE is supported by Hadassah “OFEK” research fund, ASH global research award, ISF grant
(2873/23), Israel Society of Hematology and Transfusion Medicine, and grant of the Ministry
of Innovation, Science and Technology (0007868). HJ is supported by the Golda Meir
Endowment Fund for postdoctoral fellowship. Ofer Mandelboim is supported by: The Israel
Science Foundation grant 3042/22, grant 442/18 and grant 619/23; The German -Israeli
Foundation for Scientific Research and Development Grant 1412 -414.13/2017; The Israel
Innovation Authority grant 75934; The ICRF professorship; The Yissum grant 220318; and
The MOST-DKFZ grant 3 -14931 and grant 3 14930. The funders had no role in the study
design, data collection, analysis, interpretation of data, writing of the man uscript, or the
decision to submit the article for publication.
4
Abstract
Chimeric antigen receptor (CAR) T cell therapies have revolutionized treatment of hematologic
malignancies such as lymphoma and multiple myeloma. However, their success is limited b y
high manufacturing costs, reliance on autologous T cells, variable product quality, and life -
threatening toxicities like cytokine release syndrome. In contrast, natural killer (NK) cells offer
a safer, more flexible alternative, but their clinical translation remains constrained by complex
expansion protocols and high production costs.
Here, we present a transformative approach using the human NK cell line YTS, which is
amenable to large-scale culture, genetic manipulation, and cryopreservation. By introducing a
CD19-specific CAR into YTS cells, we generate potent effector cells capable of selectively
eliminating CD19 -expressing targets. We demonstrate that CAR signaling in YTS cells
requires intracellular activation and, in certain tumor settings, is enha nced by co -stimulation
via the 2B4-CD48 pathway. Importantly, irradiation of YTS-CAR cells prevents proliferation
without compromising their cytotoxic function even after freezing and thawing. In preclinical
models, injections of irradiated YTS-CAR cells significantly reduced CD19+ tumor burden,
underscoring their therapeutic promise.
This work positions engineered YTS cells as a novel, scalable, and cost -effective “off-the-
shelf” immunotherapy platform suitable for treating refractory leukemias and lymphomas.
Future studies will be required to assess safety and to explore applicability to autoimmune
diseases and solid tumors.
5
Introduction
The expanding use of CAR-T cell therapy has revolutionized the treatment of lymphoma and
myeloma.1,2 Despite its success, this therapy still presents several limitations. A primary
constraint involves the necessity for cell production from the patient due to the MHC restriction
of T cells. This is a complex procedure that often uses T cells previously exposed to
chemotherapy, affecting their quantity a nd functionality.3,4 Additionally, CAR-T therapy can
induce severe adverse effects, some of which may be life -threatening or irreversible, such as
cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome
(ICANS).5 Over the last few years, there has been growing interest in harnessing natural killer
(NK) cells as a n alternative platform.6,7 NK cells are innate immune cells that play a pivotal
role in eliminating virally -infected and cancer cells. One key advantage of NK cells over T
cells is their lack of MHC restriction, allowing them to be used as off -the-shelf products.
Furthermore, NK cells have shown no propensity to induce CRS or ICANS,8 unlike CAR-T
cells. NK cells can be obtained from various origins, primarily from peripheral and cord blood.7
However, their production typically involves a complex and expensive process, potentially
resulting in variations in the final product administered to patients. These complexities
challenge the widespread clinical application of NK cells despite their advantages over T cells.
YTS is a subline of the NK cell line YT derived from a patient with acute lymphoblastic
lymphoma.9,10 YTS cells express CD56 dim and have NK -like features, 9–11 most notably,
cytotoxic activity in vitro and in vivo .10 Unlike other NK cell products that express a
combination of activating and inhibitory receptors, YTS cells lack inhibitory receptors 11 and
appear to possess only two functional activating receptors: 2B412 and CD28.13
YTS cells present several compelling advantages over current NK cell-based immunotherapies.
Unlike primary NK cells, which require donor -specific isolation and personalized
6
manufacturing processes that cause donor-to-donor variability, YTS cells grow robustly in
culture, are inexpensive to maintain, and can be genetically manipulated with high efficiency.
This makes them a stable, reproducible, and scalable cellular platform for immunotherapy.
Their uniform phenotype and responsiveness eliminate the variability often seen with
peripheral or cord blood–derived NK products.
In this study, we demonstrate that YTS cells engineered to express a CD19 -specific CAR
exhibit potent and selective cytotoxicity a gainst CD19+ tumor cells both in vitro and in vivo.
Moreover, we demonstrate that γ-irradiation inhibits YTS cell proliferation while maintaining
their antitumor activity, an important step toward a safe, off -the-shelf NK cell therapy.
Together, our findin gs position CAR -YTS cells as a promising and practical alternative to
current NK or CAR-T therapies, with broad translational potential.
Methods
Cell lines
The cell lines used in this study included YTS-eco (abbreviated as YTS),14 Raji, BJAB, MDA-
MB-453, HEK293T, NK-92MI and the melanoma cell lines Mel526 and A375. All lines were
cultured in-house, except the melanoma lines, kindly provided by Prof. Michal Lotem. NK-
92MI cells were kindly provided by Dr. Aladin Samara and Dr. Galit Granot (Rabin Medical
Center, Israel). Detailed culture conditions for each cell line are provided in the Supplementary
Methods.
Cloning, generation of lentivirus particles and viral transduction
7
The coding sequences of human CD48 (NM_001778.4) and CD19 (NM_001178098.2) were
cloned into the pHAGE-DsRED(−)-eGFP(+) lentiviral vector by Twist Bioscience. The CAR
sequence targeting CD19 was derived from the FMC63 -28Z construct (GenBank:
HM852952.1). Two CAR constructs were expressed using lentiviral vectors: the first, obtained
from Addgene (plas mid #135992), incorporated 4 -1BB costimulatory signaling (FMC63 -
41BB-3z-P2A-EGFP); the second, based on HM852952.1, included CD28 signaling with an
N-terminal FLAG tag in pHAGE -DsRED(−)-eGFP(+) lentiviral vector (Twist Bioscience).
Experimental outcomes were consistent across both constructs, with no significant differences
observed. For comparisons between YTS-CAR cells and other CAR-engineered cell therapies,
the FLAG-CAR-CD28 construct was used in all cell types to ensure a uniform CAR design
across platforms. Additionally, a truncated CAR variant was generated by retaining only two
amino acids from the intracellular domain of CD28 (Twist Bioscience).15
Lentiviral virions were produced via transient three-plasmid transfection. HEK293T cells were
co-transfected with the lentiviral vector containing the insert, a plasmid encoding PsPAx, and
a plasmid encoding VSV -G, using TransIT -LT1 Transfection Reagent (Mirus Bio).
Supernatants containing viral particles were collected 48 hours later and used to transduce the
target cells.
Animal experiments
All animal experiments were conducted using female NOD scid gamma (NSG) mice, ages 8-
12 weeks, bred and maintained in-house; Figure 5B was repeated once in male mice with
similar results. Mice were housed under specific pathogen-free (SPF) conditions at the Hebrew
University–Hadassah Medical School (Ein Kerem, Jerusalem). All procedures were approved
by the Institutional Animal Care and Use Committee. Tumor size was measured regularly using
calipers, and tumor volume was calculated using the formula: (length × width²) / 2.
8
Human samples
The study involving human samples was approved by the Institutional Helsinki Committee of
Hadassah Medical Center. Peripheral blood samples were collected from CLL patients into
heparinized tubes after obtaining written informed consent. PBMCs were isolated using a Ficoll
gradient, and the mononuclear cell fraction was aliquoted and cryo preserved in freezing
medium containing 90% fetal bovine serum (FBS) and 10% DMSO. Samples were thawed and
used for experiments as needed.
Statistical analysis
Statistical analysis was performed using Microsoft Excel and GraphPad Prism software
(version 7.02). Comparisons were made using Student’s t-test or one -way ANOVA, as
appropriate. A p-value < 0.05 was considered statistically significant.
Descriptions of the remaining methods are provided in Supplementary Data.
Results
YTS CAR CD19 efficiently kills CD19-positive targets
To utilize YTS cells therapeutically, we introduced a second -generation chimeric antigen
receptor (CAR) against CD19 into these cells via lentiviral transduction. The CAR construct
includes an anti -CD19 CAR linked to GFP via a P2A sequence and contains the 4-1BB and
CD3ζ signaling domains. After sorting, we obtained a homogeneous population of GFP -
positive cells (Figure 1A). We then performed an in vitro killing assay to evaluate CAR-CD19
function in YTS cells. Raji cells, which express CD19 (Supplementary Figure 1), were used
as target cells and incubated with YTS-CAR CD19 . The degree of target cell killing was
quantified using Calcein-AM labeling. While Raji cells were only modestly killed by wild-type
(WT) YTS cells, killing was significantly enhanced by YTS-CAR CD19 cells (Figure 1B). In
9
parallel, the CD19⁺ B -cell line BJAB 16 was also efficiently and specifically killed by YTS -
CAR CD19 cells (Figure 1B ). Similar results were observed when chronic lymphocytic
leukemia (CLL) cells , which also express CD19 ( Supplementary Figure 1), were used as
targets, as assessed by flow cytometry (Figure 1C).
Next, we aimed to verify that the enhanced killing observed in YTS-CAR CD19 cells was due
to the activity of the introduced CAR and not merely improved engagement between YTS cells
and the target cells. To achieve this, we expressed another CAR construct in YTS cells lacking
the intracellular signaling domain, which has previously been shown to retain antigen-specific
tumor-targeting capability 15 (see Methods). As before, we confirmed the transduction
efficiency by examining GFP expression levels ( Figure 1D). However, the truncated CAR
construct did not demonstrate enhanced killing capability (Figure 1E). Therefore, we conclude
that the intracellular signaling domain of the CAR is essential for its function in YTS cells.
We next evaluated degranulation and cytokine production by YTS -CAR CD19 cells. YTS -
CAR CD19 cells exhibited relatively high baseline CD107a expression, consistent with the
activated phenotype of this cell line. However, co -culture with Raji target cel ls resulted in a
further increase in CD107a expression ( Supplementary Figure 2A). In addition, YTS-CAR
CD19 cells expressed high baseline levels of granzyme B, interferon -γ (IFN -γ), and tumor
necrosis factor -α (TNF -α), with further increases in granzyme B and IFN -γ expression
following co -culture with Raji cells (Supplementary Figure 2B ). We also assessed the
expression of inhibitory receptors to evaluate the exhaustion profile of YTS-CAR cells. While
TIM-3 and LAG -3 were notably absent, PD -1 was expressed at baseline and further
upregulated following co -culture with target cells (Supplementary Figure 2C ). These
findings indicate that, while YTS -CAR CD19 cells display basal activation, their effector
function is further augmented upon target engagement.
10
We also investigated the potential of YTS cells to kill solid cancer cells. To test this, we
expressed CD19 in the melanoma cell lines Mel526 and A375 ( Figure 2A,B ). YTS cells
expressing the CAR against CD19 were able to specifically kill melanoma cells ex pressing
CD19 ( Figure 2C,D ). In contrast, YTS cells expressing a truncated CAR lacking the
intracellular signaling domain failed to mediate target cell killing, confirming that cytotoxicity
depends on CAR signaling (Figure 2C,D). This finding highlights the potential of YTS-CAR
cells to recognize and kill non-hematologic cells when they express the relevant target antigen.
The 2B4 -CD48 interaction is important for the killing of hematologic tumors by YTS
CAR CD19
Previously, only two receptors, 2B4 12 and CD28, 13 were identified as active in YTS cells.
However, the basal activity of YTS cells against target cells expressing the ligands for these
receptors is low. Raji cells, which express CD48 (the ligand for 2B4) as well as CD80 and
CD86 (the ligands for CD28) (Supplementary Figure 1), were minimally killed by YTS cells
(Figure 1B). Likewise, primary dendritic cells (DCs), which also express CD80 and CD86,
were largely resistant to YTS-mediated killing (Supplementary Figure 3). Despite this, 2B4
and CD28 may still contribute to YTS -mediated cytotoxicity when YTS cells express CAR
against CD19. To assess the importance of these receptors in CAR -CD19-mediated
cytotoxicity, we generated MDA -MB-453 transfectants expressing CD19, CD48, or both
(Supplementary Figure 4) and tested their susceptibility to YTS expressing CAR CD19 cells
in a cytotoxicity assay (Figure 3A). While the expression of CD19 or CD48 alone did not
trigger significant killing, the co-expression of both ligands resulted in substantial cytotoxicity
(Figure 3A). We also evaluated the expression of CD48, CD80, and CD86 in the melanoma
cell lines Mel526 and A357. Despite these cell lines bei ng efficiently killed by YTS cells
expressing CAR CD19 (Figure 2), they exhibited low or minimal expression of these ligands
(Supplementary Figure 1).
11
To further investigate the roles of these receptors, we either disrupted or blocked them in YTS
cells. Using CRISPR, we knocked out 2B4 in YTS cells and subsequently overexpressed CAR
targeting CD19 (Figure 3B). The deletion of 2B4 abolished the enhanced killing observed in
CAR-expressing YTS cells when incubated with Raji cells (Figure 3C). To assess the role of
CD28, we used a blocking antibody that has previously been shown to reduce YTS -mediated
killing of 721.221 cells (13 and Supplementary Figure 5). We applied this antibody in a
cytotoxicity assay using YTS cells expressing CAR against CD19 and Raji cells, which express
CD28 ligands (Supplementary Figure 1). However, blocking CD28 did not affect the killing
of Raji cells by CAR-expressing YTS cells (Figure 3D). In conclusion, while CD28 does not
appear to contribute to CAR -mediated killing by YTS cells, the 2B4 -CD48 interaction may
play a role in certain cell types, such as MDA -MB-453 and Raji. However, this interaction
appears dispensable in melanoma cells , where robust cytotoxicity was observed despite the
absence of the 2B4 ligand CD48 and the CD28 ligands CD80 and CD86.
Ensuring Safety and Efficacy: Irradiation of YTS -CAR Cells Retains Anti -Tumor
Activity While Preventing Proliferation in vitro and in vivo
To use YTS cells therapeutically, they must be irradiated before injection into patients to
prevent their proliferation. It is therefore essential to confirm that irradi ation effectively
prevents cell proliferation while preserving their cytotoxic activity. To evaluate the effects of
irradiation on YTS cells in vitro, we employed several methods. First, we quantified the number
of viable cells after exposing them to vario us irradiation doses. A minimum dose of 1000 rad
effectively inhibited cell proliferation, as indicated by a reduction in viable cell numbers
(Figure 4A and Supplementary Figure 6A). Th is observation was supported by an MTT
assay, which demonstrated a decrease in metabolic activity beginning at doses as low as 1000
rad (Figure 4B and Supplementary Figure 6B). We also tested irradiated (2500 rad) YTS -
12
CAR cells following a freeze–thaw cycle and confirmed that they did not regain proliferative
capacity (Supplementary Figure 6C).
To further assess proliferation, cells were labeled with a CellTrace dye, confirming that a 1000
rad dose was sufficient to prevent proliferation (Figure 4C). To investigate the in vivo impact
of YTS cell irradiation, we subcutaneously injected NSG mice with CAR -CD19–expressing
YTS cells exposed to varying radiation doses. Tumor growth was monitored over a follow-up
period of 5 months. Mice receiving YTS cells that were non-irradiated or irradiated at 1000 rad
developed tumors, whereas those injected with cells irradiated at 2000 rad or higher showed
no tumor growth throughout a 5 -month observation period (Figure 4D). Based on the results
of both in vitro and in vivo experiments, we determined that a 2500 rad irradiation dose is
effective in preventing cell proliferation. Next, we evaluated whether the cytotoxic activity of
YTS cells expressing CAR was retained after irradiation at 2500 rad. Additionally, we froze
the cells post-irradiation to simulate how these engineered YTS cells could be administered to
patients. A cytotoxicity assay was performed, in which irradiated YTS cells, both with and
without CAR targeting CD19, were incubated with Raji cells. The assay sho wed that YTS -
CAR CD19 cells maintained their cytotoxic activity after irradiation and freezing (Figure 4E).
Irradiated YTS -CAR cells retained cytotoxic activity for up to 6 days, with only a mild
reduction over time (Supplementary Figure 6D). In addition, irradiated YTS-CAR cells were
detectable in the circulation of mice for up to 24 hours after infusion (Supplementary Figure
6E). Since YTS cells harbor EBV consistent with latent infection 10,17, we further assessed
whether EBV could be transferred to recipient cells. Co -culture experiments with HEK293T
cells showed no evidence of EBV infection (Supplementary Figure 6F).
Comparison of YTS cells with other CAR -based cellular therapies and in vivo tumor
control by irradiated YTS-CAR CD19 cells
13
We next compared the cytotoxic activity of YTS-CAR CD19 cells with other CAR-expressing
cellular products. NK -92 is the most widely used NK cell line in clinical studies; however,
these cells proliferate more slowly than YTS cells ( Supplementary Figure 7A). To enable a
direct comparison, YTS cells, NK-92 cells, and primary T cells were transduced with the same
FLAG-tagged CAR construct containing a CD28 co-stimulatory domain (Figure 5A, left, top).
Cytotoxicity assays were performed using equal numbers of Raji target cells and CAR-positive
effector cells. To mimic clinical use, frozen-thawed effector cells were tested both before and
after irradiation at the recommended dose for each cell type (CAR-T cells were not irradiated).
YTS-CAR CD19 cells exhibited cytotoxic activity comparable to NK-92 CAR cells under both
conditions and greater than that observed with primary CAR -T cells expressing the same
construct (Figure 5A, left). We next compared cytokine secretion by the CAR-expressing NK
cell lines following co-culture with target cells. YTS -CAR CD19 cells secreted higher levels
of interferon -γ (IFN -γ) compared with NK -92 CAR cells after incubation with Raji cells
(Figure 5A, right).
Finally, to assess the therapeutic function of YTS cells expressing CAR CD19 in vivo, we used
a tumor model in which 7x106 Raji cells were injected subcutaneously into NSG mice. Once
tumors became palpable, we injected 20x106 YTS CAR CD19 cells, which had been irradiated
at 2500 rad and frozen, approximately twice a week for three weeks. As controls, we used non-
transduced YTS cells or PBS. Tumor size was evaluated, and injection of YTS CAR CD19
cells resulted in a significant reduction in tumor size (~50%) ( Figure 5B, Supplementary
Figure 7B).
Discussion
Here, we demonstrate that genetically engineered YTS cells represent a promising approach to
cellular therapy. As a proof of concept, we engineered YTS cells to express a CAR targeting
14
CD19, which showed enhanced activity both in vitro against CD19+ cell line and primary CLL
cells, and in vivo.
NK cells offer several advantages over T cells for immunotherapy, including reduced adverse
effects such as cytokine release syndrome and immune-effector cell-associated neurotoxicity.6
Unlike T cells, NK cells are not restricted by major histocompatibility complex (MHC)
compatibility, allowing their use a readily available off-the-shelf therapy. Additionally, NK
cells exhibit natural cytotoxicity against tumor cells. Despite these advan tages, NK cells face
significant challenges as a therapeutic option. They represent a rare cell population, have a
shorter lifespan than T cells in vivo, and are not easily manipulated. Various approaches have
been developed to harness NK cells for immunot herapy. These include isolating primary NK
cells from peripheral blood with modifications such as treatment with nicotinamide 18 or
stimulation with IL -12, IL-15, and IL -18 to initiate a memory -like program.19 Other sources
include NK cells directly derived from cord blood ,8,20 those matured from cord blood stem
cells,21 or NK cells derived from human induced pluripotent stem cells (iPSCs) .22 Another
notable source of NK cells is the NK-92 cell line, which has been investigated as a therapeutic
option for various tumors with modifications such as the e xpression of different chimeric
antigen receptors (CARs) .23 NK-92-based CAR therapies have been evaluated in several
clinical trials, including CARs targeting HER2 24 and CD33. 25 However, despite their
therapeutic potential, NK -92 cells present practical limitations: they proliferate relatively
slowly ( Supplementary Figure 7 A), require specialized culture conditions, 26 and can be
challenging to genetically modify (personal observation), all of which may complicate clinical
translation. In addition, the broader receptor repertoire of NK-92 cells compared to YTS cells27
may increase the risk of off -target effects. In our study, YTS -CAR CD19 cells demonstrated
cytotoxic activity comparable to NK -92 cells expressing the same CAR construct, while
exhibiting higher secretion of interferon -γ (IFN -γ). Increased IFN -γ productio n has been
15
associated with improved anti -tumor responses, including in CAR -T cell therapies, 28,29 and
especially in the case of solid cancers.30
YTS cells offer several advantages over other NK cell sources. They are easy to culture, readily
transfected to express additional receptors, and naturally lack inhibitory receptors. Unlike most
NK cells, which rely on a delicate balance of complex activating and inhibitory receptors ,31
YTS cells have only a few active receptors.12,13 This limited receptor repertoire minimizes off-
target effects while allowing for precise targeting of antigens. Additionally, unlike CAR -T
cells, YTS cells do not require specialized collection processes and can be administered
immediately to patients, eliminating production delays. We did not directly assess the potential
of YTS cells to induce CRS,32 as this is not reliably modeled in NSG mice. However, clinically
significant CRS or neurotoxicity has not been observed in studies using other NK cell -based
platforms.8,33 These features make YTS cells an accessible and potent NK cell source,
addressing key limitations of CAR -T therapies, particularly technical challenges and high
costs.34–36 To address safety, we performed a series of in vitro and in vivo experiments to
determine the irradiation dose required to prevent cellular proliferation. Irradiation at ≥2 000
rad resulted in irreversible loss of proliferative capacity and progressive cell death. In vivo,
irradiated YTS-CAR cells were injected into immunodeficient mice and monitored for up to 5
months, with no evidence of tumor formation. Although irradiated cells did not exhibit
proliferative capacity in vitro or in vivo , additional analyses of genomic instability or
transformation markers could provide further safety information and should be evaluated in
future studies. In addition, YTS cells, as a subclone of the YT NK-like lymphoma line, harbor
Epstein–Barr virus (EBV) consistent with latent infection. 10,17 The theoretical risk associated
with EBV carriage is mitigated by pre -infusion irradiation, which abolishes proliferative
capacity, as well as by the intended short-lived, non-engrafting use of these cells, as confirmed
by our in vitro experiments. Notably, the NK -92 cell line has also been reported to contain
16
EBV DNA sequences, 37–39 and irradiated NK -92 cells have been administered clinically
without evidence of EBV-related safety concerns. In terms of treatment efficacy, although we
observed an approximately 50% reduction in tumor size, our model does not fully recapitulate
lymphoma, as tumor cells were implanted subcutaneously. Importantly, YTS cells also secrete
high levels of IFN -γ, a po tent immunomodulator whose full therapeutic potential is likely
underestimated in this immunodeficient mouse model. While only a single CD19⁺ tumor model
was evaluated in vivo, additional CD19⁺ target cell s were shown to be susceptible to YTS -
CAR-mediated killing in vitro , supporting the broader cytotoxic potential of these cells.
Finally, we have not compared YTS -CAR to CAR -T in vivo , as both platforms have
fundamentally different kinetics, which would make a direct comparison difficult to interpret.
Mechanistically, we demonstrate that CAR activity against CD19 relies on intracellular
signaling, indicating that its effectiveness is not solely due to CAR -target cell engagement.
Additionally, we show that for some tumors the CAR’s function depends on th e 2B4-CD48
axis, suggesting that this pathway should be evaluated in specific tumor types. It remains
unclear why melanoma cells, which were resistant to YTS-mediated killing at baseline, became
susceptible to YTS -CAR-mediated cytotoxicity upon CD19 expres sion, unlike MDA cells.
This differential response may be due to the expression of a co-stimulatory ligand by melanoma
cells that engages a receptor on YTS cells, providing a secondary activation signal effective
only with CAR engagement.
We assessed the effect of CD19-targeted CARs in YTS cells, as this is the most widely studied
CAR. However, it is plausible that other CARs may also be effective in YTS cells. Moreover,
additional elements can be transduced into YTS cells, positioning them as a versatile platform
for diverse receptor expression and expanded therapeutic potential. However, although the
irradiated NK -92 cell line has been used in clinical trials, demonstrating the safety of this
approach, the safety of irradiated YTS cells will need to be carefully evaluated in future studies.
17
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20
Figures legends
Figure 1. YTS expressing CAR against CD19. A) FACS verification of CAR CD19
expression in YTS cells; (B, C) Killing experiments: tumor cells (Raji and BJAB, B or primary
CLL cells, C) were incubated with YTS cells expressing CAR against CD19 or an empty
vector. For B, E:T 20:1, repeated four times; For C, E:T 20:1, repeated in two different patients.
D) FACS verification of CAR CD19 with truncated intra -cellular domain expressed in YTS
cells. E) Killing experiment: YTS cells expressing CAR CD19, truncated C AR CD19 or an
empty vector, were incubated with Raji cells, E:T 20:1, repeated two times. Killing experiments
were performed in at least five technical repeats and target cells were labeled with Calcein AM.
***, P<0.001, ****, P<0.0001, Student’s t-test. CLL – chronic lymphocytic leukemia, E:T –
Effector: Target.
Figure 2. Activity of YTS cells against melanoma cell lines. (A, B) Expression of CD19 in
melanoma cell lines: Mel526 (left) and A357 (right). Cells were stained by FACS for CD19.
(C, D) Killing assays: melanoma cell lines (Mel526, C; A357, D) with and without CD19
expression were labeled with Calcein AM and then incubated with YTS cells expressing CAR
against CD19, a truncated CAR lacking the intracellular signaling domain, or an empty vector.
E:T 20:1. One experiment is presented out of two performed, the killing experiments were
performed in six replicates. ***, P<0.001, Student’s t-test or ANNOVA with multiple
comparisons.
21
Figure 3. Interaction of CAR against CD19 with other receptors in YTS cells. (A) Killing
assays with MDA-MB-453 transfectants expressing empty vector, CD19, CD48 or both CD19
and CD48 were incubated with YTS WT or YTS expressing CAR against CD19. (B, C) FACS
analysis showing 2B4 expression ( left) and GFP expression ( right), where GFP serves as a
reporter for CAR expression, in YTS cells with 2B4 KO expressing CAR against CD19. (C)
Killing assay: YTS expressing CAR against CD19 or YTS expressing CAR against CD19 with
2B4 KO were incubated with Raji cells, E:T 20:1. (D) Killing assay: YTS cells expressing
CAR against CD19 were pre-incubated with either a control antibody or an anti-CD28 antibody
before being incubated with target cells at an E:T ratio of 20:1. All experiments were performed
at least twice, killing experiments were performed in six replicates and target cells were labeled
with Calcein AM, ***, P<0.001, Student’s t-test.
Figure 4. Safety and efficiency of irradiated YTS cells . A-C) YTS cells expressing CAR
against CD19 were non-irradiated or irradiated at different levels and then subjected for various
viability and proliferation tests along different time points (repeated in two independent
experiments): (A) Viability test, where the number of viable cells was detected by FACS; (B)
MTT assay to assess the metabolic activity of the cells, and (C) Proliferation assay to test the
proliferation capacity of the cells using CellTrace. (D) 7x106 YTS CAR CD19 cells were
irradiated at various doses and subsequently injected subcutaneously into NSG mice. Tumor
development was monitored over time. Each group included four mice. The results were
repeated in another independent experiment. (E) YTS cell s expressing either a CAR against
CD19 or an empty vector were irradiated with 2500 rad or left non-irradiated, then frozen and
later used in a killing assay against Raji cells. The degree of killing was assessed by FACS at
multiple time points over 48 hours. Representative raw flow cytometry plots of Raji cells after
22
24 hours are shown on the left. E:T 20:1. ***, P<0.001, ANOVA test with multiple
comparisons (A, B, E).
Figure 5. Comparison of YTS -CAR CD19 cells with other CAR -based effectors and in
vivo activity of YTS -CAR CD19. (A) Left: YTS cells, NK -92MI cells or primary T cells
expressing a CD19-targeted CAR (YTS -CAR, NK-92 CAR, and CAR -T, respectively) were
co-cultured with Raji target cells at an effector-to-target (E:T) ratio of 10:1. All CAR constructs
were identical and included a FLAG tag to enable detection of CAR expression. Frozen-thawed
effector cells were either left untreated or irradiated prior to the assay (YTS: 2500 rad; NK-92:
1000 rad). CAR-T cells were not irradiated. For CAR-expressing cells, input cell numbers was
adjusted to ensure equivalent numbers of CAR-positive cells across conditions, based on FLAG
expression (shown above). Cytotoxicity was assessed by flow cytometry after 3 hours of
incubation. Data shown are representative of two independent experiments with similar results.
Right: IFN-γ secretion by YTS -CAR and NK-92 CAR CD19 cells following co -culture with
Raji target cells at an E:T ratio of 5:1 for 24 hours. Supernatants were collected and analyzed
by ELISA. (B) In vivo assessment of YTS-CAR CD19 cells in tumor-bearing immunodeficient
mice. 7x106 Raji cells were injected subcutaneously into immunodeficient mice (indicated by
a filled arrow). Once tumors became palpable, mice were treated with 20x10 6 YTS cells
expressing CAR-CD19, untransduced YTS cells, or PBS, injected twice weekly (indicated by
hollow arrows). YTS cells were irradiated at 2500 rad and cryopreserved prior to injection.
Tumor volumes were monitored throughout the experiment. Similar results were observed in
two additional independent experiments (one using cells irradiated at 1000 rad and another
using cells irradiated at 2500 rad administered approximately three times per week). * P<0.05,
** P<0.01, **** P<0.0001, ANOVA with multiple comparisons (A), Student’s t-test (B).
1
Supplementary Methods
Cell lines
Raji and YTS cells were cultured in RPMI -1640 medium (Sigma-Aldrich), while MDA-MB-
453 and HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM,
Sigma-Aldrich). Culture media were supplemented with 10% heat -inactivated fetal bovine
serum (FBS, Sigma-Aldrich), 1 mM sodium pyruvate (Biological Industries), 2 mM glutamine
(Biological Industries), non-essential amino acids (Biological Industries), 100 U/mL penicillin
(Biological Industries), and 0.1 mg/mL streptomycin (Biological Industries). All cells were
maintained at 37°C in a humidified incubator with 5% CO₂. The melanoma cell lines Mel526
and A375 were cultured in a mixed medium comprising 75% DMEM, 25% RPMI-1640, and
8.75% Ham's F -12 (Biowest), supplemented with 10% heat -inactivated FBS, 1.25% HEPES
buffer (Biological Industries), 1.25% 100 mM glutamine, and 1.25% penicillin -streptomycin.
NK-92MI cells were maintained in α-MEM without ribonucleosides (low glucose) (Sartorius)
supplemented with 12.5% heat-inactivated FBS, 12.5% heat-inactivated horse serum (Merck),
2 mM L-glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, and penicillin (100
U/mL)–streptomycin (0.1 mg/mL). The medium was further supplemented with 0.2 mM myo-
inositol (Merck), 0.1 mM 2-mercaptoethanol (Merck), and 0.02 mM folic acid (Merck).
Antibodies
Antibodies used for FACS analysis included: APC anti -human CD19 (BioLegend; cat . no.
302212), APC anti-human CD48 (BioLegend; cat. no. 336714), APC anti-human CD244 (2B4)
(BioLegend; cat. no. 329512), APC anti-human CD80 (BioLegend; cat. no. 305220), APC anti-
human CD86 (BioLegend; cat. no. 374208), PE anti-human CD352 (NTB-A) (BioLegend; cat.
no. 317208), Anti-Human CD28 (BD; cat. no. BD348040).
2
Isotype control antibodies used: APC Mouse IgG1, κ (BioLegend; cat No. 400120) and PE
Mouse IgG1, κ (BioLegend; cat no. 400112).
FACS staining
For FACS staining, cells were counted and plated in 96 -well U-bottom plates at a density of
100,000 cells per well. Cells were incubated with antibodies under the following conditions:
Unconjugated primary antibodies were added at 0.2 µg per well and incub ated on ice for 1
hour. After incubation, cells were centrifuged at 1600 rpm for 5 minutes at 4°C, and a
fluorophore-conjugated secondary anti -mouse antibody was added at a 1:200 dilution. For
directly conjugated antibodies, staining was performed using a 1:200 dilution with a 30-minute
incubation on ice. Following staining, cells were either analyzed immediately or fixed with 1%
paraformaldehyde (PFA) for later acquisition. FACS analysis was performed using a DxFLEX
or CytoFLEX flow cytometer.
Lentivirus production
Lentiviral virions were produced via transient three-plasmid transfection. HEK293T cells were
co-transfected with the lentiviral transfer vector encoding the CAR construct, a packaging
plasmid (psPAX2), and a plasmid encoding VSV-G, using TransIT-LT1 Transfection Reagent
(Mirus Bio) according to the manufacturer’s instructions. Supernatants containing viral
particles were collected 48 hours post-transfection, filtered through a 0.45 μm filter, and used
immediately or stored at −70°C until use.
Intracellular cytokine and cytotoxic molecule staining
For assessment of cytokine production and cytotoxic effector molecules, YTS-CAR cells (2 ×
10⁶) were co-cultured with Raji target cells (1 × 10⁵) at a 20:1 effector -to-target (E:T) ratio in
RPMI complete medium (500 µL + 500 µL). Control wells contained YT S-CAR cells alone.
3
Cells were incubated for 3 hours at 37°C in the presence of GolgiPlug (1:1000; BD
Biosciences, cat. no. 554714).
Following incubation, 200 µL of cells were collected, washed with FACS buffer, and stained
for surface CD56-PE (BioLegend, cat. no. 318306; 1:200 dilution) for 30 minutes on ice. Cells
were then fixed and permeabilized using BD Cytofix/Cytoperm solution (cat. no. BD554714,
BD Biosciences) for 30 minutes at room temperature. After washing with Perm/Wash buffer,
intracellular staining was performed using antibodies against granzyme B -APC (BioLegend,
cat. no. 396408), IFN-γ-BV650 (BioLegend, cat. no. 502538), and TNF-α-BV785 (BioLegend,
cat. no. 502948), each diluted 1:100 in Perm/Wash buffer. Cells were washed and analyzed by
a DxFLEX flow cytometer (Beckman Coulter).
Exhaustion marker analysis
To evaluate expression of exhaustion markers, YTS-CAR cells (2 × 10⁶) were co-cultured with
Raji cells (1 × 10⁵) at a 20:1 E:T ratio in RPMI complete medium for 24 hours at 37°C.
Following incubation, 200 µL of cells were collected, washed, and stained wit h the following
antibodies diluted 1:100 in FACS buffer for 30 minutes on ice: TIM-3-PE (Thermo Fisher, cat.
no. 12 -3109-42), PD -1-PC5.5 (Beckman Coulter, cat. no. B36123), and LAG -3-AF647
(Thermo Fisher, cat. no. 51 -2239-42). Cells were washed and analyze d by flow cytometry
using a DxFLEX instrument.
CD107a degranulation assay
Degranulation was assessed by measuring surface CD107a expression. YTS -CAR cells (1 ×
10⁵) were co-cultured with Raji target cells (1 × 10⁵) at a 1:1 ratio in 200 µL RPMI complete
medium. Anti-CD107a-APC (BioLegend, cat. no. 328620) and CD56-PE (BioLegend, cat. no.
318306) were added at the beginning of the incubation (0.2 µg per well). Cells were incubated
for 2 hours at 37°C, washed with FACS buffer, and analyzed by flow cytometry on a DxFLEX
instrument.
4
Viral transduction
For transduction, YTS and NK-92MI cells were resuspended in viral supernatant and seeded
in 96-well U-bottom plates, followed by spinoculation at 1800 rpm for 90 minutes at 32°C.
Cells were then incubated overnight at 37°C and cultured under standard conditions. CAR -
expressing cells were purified by FACS (SH800, Sony) based on GFP or FLAG expression.
The gating strategy included exclusion of debris based on forward and side scatter, and
selection of GFP⁺ or FLAG⁺ live cells. Sorting yielded a homogeneous CAR -positive
population that was used for all downstream experiments.
Cytotoxicity assay
The in vitro cytotoxic activity of YTS eco cells against various target cells was assessed either
by Calcein -AM-labeled target cells (ThermoFisher Scientific , cat. no. C1430 ) or FACS.
Although the absolute levels of cytotoxicity were not identical across methods, they provided
a reliable basis for comparison between the various cell platforms. Cytotoxicity was evaluated
at multiple time points, including 3, 24, and 48 hours, depending on the specific experiment.
For the Calcein-AM assay , target cells (3 × 10⁶) were counted, washed with RPMI, and
resuspended in Calcein-AM (1 mg/mL) diluted at a 1:200 ratio. The cells were then incubated
at 37°C for 30 minutes , centrifuged, washed with RPMI, and resuspended in RPMI+. T en
thousand cells were plated per well in a 96 -well U -bottom plate. For standard cytotoxicity
assays, target cells were resuspended in 100 µL RPMI+. For ADCC experiments, target cells
were resuspended in 50 µL RPMI+, incubated with antibodies at 0.5 µg/well (in 50 µL volume)
for 1 hour on ice, and then mixed with YTS cells at a predetermined effector -to-target (E: T)
ratio in an additional 100 µL. For CD28 blocking, YTS cells were pre -incubated with 0.5 µg
of anti-CD28 antibody (clone L293, BD Biosciences, cat. no. 348040) for 1 hour on ice. Each
experiment was performed in quadruplicate. Plates were incubated at 37°C for 3 hours,
5
centrifuged, and 100 µL of the supernatant was transferred to black, flat-bottom 96-well plates
(Greiner) for fluorescence measurement using either a Tecan plate reader or a GloMax Explorer
(Promega). Spontaneous release controls contained target cells incubated in medium only, and
maximum lysis controls were treated with 50 nM NaOH. Percent specific lysis was calculated
using the formula: % Cytotoxicity = [(experimental release – spontaneous release) / (maximum
release – spontaneous release)] × 100.
For assessment of cytotoxicity by flow cytometry, YTS CAR -CD19 (1 × 10⁶ cells/mL) were
co-cultured with Raji target (1 × 10⁵ cells/mL) at an E:T ratio of 10:1 for 3 hours at 37°C.
Following incubation, cells were collected and stained with Zombie NIR viability dye (diluted
1:1000), then with anti-CD19 (APC) and anti-CD56 (PE) antibodies (diluted 1:200). Live cell
counts were normalized to control wells, which contained target cells alone in medium.
Production of CAR T cells and NK-92 CAR
We cloned CAR -CD19 with a CD28 costimulatory domain into an MSCV retroviral vector
with puromycin resistance, adding an N-terminal FLAG tag. Retroviral particles were initially
produced using EcoPhoenix cells, and the resulting supernatant was used to transduce PG13
cells. Puromyci n-resistant PG13 clones were selected using puromycin (InvivoGen),
establishing stable producers of the retrovirus. Peripheral blood mononuclear cells (PBMCs)
were isolated from a healthy donor by Ficoll density gradient centrifugation, and stimulated for
48 hours with anti -CD3 (50 ng/mL, Miltenyi Biotec) and IL -2 (300 IU/mL, Novartis).
Subsequently, PBMCs were transduced with viral supernatant collected from the PG13
producer cells. Transduction efficiency was confirmed by FACS, with approximately 50% of
cells expressing the CAR. The same retroviral system was used to transduce NK -92 cells to
generate NK-92 CAR cells. In parallel, the identical FLAG-tagged CAR sequence was cloned
into a lentiviral vector and used to generate YTS -CAR CD19 FLAG cells, as described in the
6
main Methods section. CAR -positive NK-92 CAR and YTS-CAR cells were enriched using
anti-FLAG magnetic selection . Briefly, cells were incubated with anti -FLAG antibody
(BioLegend, cat. no. 637323) followed by magnetic labeling and separation using the Miltenyi
Biotec system (cat. no. 130-042-401) according to the manufacturer’s instructions.
Cell Irradiation and Assessment of Cell Proliferation
Cells were irradiated at varying doses using the MultiRad 350 X -ray irradiator (Precision X -
Ray). To assess the effects of irradiation on cell proliferation and viability, we employed the
following methods:
Thiazolyl Blue Tetrazolium Bromide (MTT) assay
Fifty milligrams of MTT (Sigma -Aldrich) were dissolved in 10 mL of 1× PBS (Biological
Industries) and filtered using a 0.22 µm filter (Millipore). Cells were counted and plated at a
density of 3 × 10⁵ cells/mL. They were then exposed to escalating doses of irradiation: 1000,
2000, 3000, and 6000 rad. Each day, 100 µL of cell suspension was transferred into 96 -well
U-bottom plates, followed by the addition of 10 µL MTT stock solution per well. The plate
was gently mixed by pipetting and incubated at 37°C for 3 hours. After incubation, the plate
was centrifuged at 1600 rpm for 5 minutes to pellet the cells, and the medium was carefully
removed. DMSO (100 µL) was added to each well, and the cells were thoroughly resuspended.
The plate was then incubated at 37°C for 10 minutes, and 90 µL of each well’s contents was
transferred to flat -bottom 96 -well plates. Absorbance was measured at 490 nm using a
microplate reader (BioTek).
CellTrace proliferation assay
7
Fifteen million cells (1.5 × 10⁷) were stained with 0.2 µM CellTrace ™ Far Red Cell
Proliferation dye (Invitrogen) in a total volume of 1.5 mL, following the manufacturer’s
instructions. Stained cells were diluted to a final concentration of 3 × 10⁵ cells/mL and
irradiated at 1000, 2000, 3000, and 6000 rad. Daily, 500 µL samples were collected and fixed
in 1% paraformaldehyde (PFA). Samples were analyzed on the same day using a DxFLEX
Flow Cytometer (Beckman Coulter).
Viability testing
Cell viability was assessed using the Zombie NIR ™ Fixable Viability Kit (BioLegend),
according to the manufacturer’s protocol. Cells were counted and diluted to 3 × 10⁵ cells/mL,
then exposed to the same irradiation doses: 1000, 2000, 3000, and 6000 rad. Each day, 200 µL
of the culture was transferred to 96-well U-bottom plates and stained with the viability dye at
1:1000 dilution. All samples were analyzed on the same day using the DxFLEX Flow
Cytometer (Beckman Coulter).
IFN-γ ELISA
IFN-γ secretion was quantified using a sandwich ELISA with a biotin –streptavidin–HRP
detection system. ELISA plates were coated overnight at 4°C with anti–IFN-γ capture antibody
(0.2 µg/ml, BioLegend, cat. no. 502402), washed with 0.05% Tween 20 in phosphate-buffered
saline (PBST), and blocked with 1% BSA for 2 hours at 37°C. NK-92 or YTS expressing CAR
CD19 effector cells were co-cultured with Raji target cells at an effector -to-target ratio of 5:1
for 24 hours, after which supernatants were collected and centrifuged. Supernatants (200
µl/well) were added to the plates and incubated for 2 hours at 37°C. Plates were then incubated
with biotinylated anti–IFN-γ detection antibody (0.1 µg/ml, BioLegend, cat. no. 502504) for 1
hour at room temperature, followed by streptavidin–HRP for 30 minutes. Signal was developed
8
using TMB substrate, and absorbance was measured at 450 nm using a GloMax microplate
reader. Experiments were performed independently four times.
EBV Transfer assessment
To assess EBV transfer, YTS -CAR CD19 cells were added to HEK293T cells at an effector -
to-target ratio of 5:1 and co-cultured for 24 hours at 37°C. Following incubation, non-adherent
YTS cells were removed by repeated PBS washes, and the remaining adherent HEK293T cells
were cultured in fresh medium for an additional 5 days at 37°C. Total genomic DNA was then
extracted from the HEK293T cells using the DNeasy Blood & Tissue Kit (Qiagen, cat. no.
69504) according to the manufacturer’s instructions. The presence of EBV DNA was assessed
by qPCR using primers specific for the BALF5 gene.
Knockout of 2B4
gRNA targeting the 2B4 receptor (CD244) (5’ -CACCGCTGCTCCTCAAGGTGTATCA-3’)
was cloned into the lentiGuide-Puro plasmid (Addgene #52963) using Esp3I, transformed into
E. coli, and verified by Sanger sequencing. Plasmid DNA was purified using the ZymoPURE
II Maxiprep Kit. Lentivirus was produced by transfecting HEK293T cells (1.25 × 10⁶
cells/well) with the gRNA plasmid, psPAX2 (Addgene #12260), and pCMV-VSV-G (Addgene
#8454) using TransIT-LT1. Viral supernatant was collected 48 hours later, filtered, and stored.
Cas9-expressing YTS cells (5 × 10⁵ cells/mL), generated in-house, were infected with the virus
in the presence of 6 μg/mL polybrene, followed by spinfection (1,800 rpm, 90 min, RT).
Medium was replaced two days post-infection.
Dendritic cell extraction
Monocyte-derived dendritic cells (DCs) were generated from peripheral blood mononuclear
cells (PBMCs) collected from a healthy donor. PBMCs were washed, seeded in 35 mm culture
plates, and incubated at 37°C with 5% CO2 to allow monocyte adherence. Non-adherent cells
9
were removed, and monocytes were cultured in media containing 1% autologous plasma, 1,000
U/mL GM-CSF, and 1,000 U/mL IL -4. Every 2 days, 0.25 mL of media was replaced with
0.35 mL of fresh media containing plasma, GM -CSF (500 U/mL), and IL -4 (500 U/mL). O n
day 6, immature DCs were harvested by trypsinization and scraping. To generate mature DCs,
on day 6, cells were stimulated with 10 ng/mL LPS and incubated for an additional 24 hours.
Morphological changes (increased size and pseudopods) confirmed activation. Cells were then
detached using trypsin-EDTA and scraped for further analysis.
10
Supplementary Figures
Supplementary Figure 1. Flow cytometry analysis of CD19, CD48, CD80, and CD86
expression across various cell lines. Data shown are representative of two independent
experiments.
11
Supplementary Figure 2. Intracellular cytokine, cytotoxic molecule, and exhaustion
marker staining of YTS -CAR CD19 cells. YTS-CAR CD19 cells were analyzed in the
presence or absence of Raji target cells for CD107a expression (A), intracellular granzyme B
and cytokine production (B), and exhaustion marker expression (C). Effector-to-target (E:T)
ratios were 10:1 for panel A and 20:1 for panels B and C. Panels A and B were performed in
triplicate. Data shown are from one representative experiment out of two performed. ** P<
0.01, *** P < 0.001, **** < P 0.0001, Student’s t-test.
12
Supplementary Figure 3. Cytotoxicity of YTS cells against dendritic cells.
(A, B) FACS analysis of CD80 and CD86 expression in immature (A) and mature (B) dendritic
cells. (C, D) Cytotoxicity assays using Calcein -AM–labeled immature (C) and mature (D)
dendritic cells co-cultured with YTS cells expressing either an empty vector or a CD19-specific
CAR. Effector-to-target (E:T) ratio was 20:1.
13
Supplementary Figure 4. Transfectants of MDA-MB-453 cells expressing CD19 and
CD48. FACS analysis of MDA-MB-453 cells showing expression of CD19, CD48, or both.
Illustrations in the top row were created with BioRender.com.
14
Supplementary Figure 5. Cytotoxicity of CAR-expressing YTS cells against 721.221 cells
with CD28 blockade . YTS cells expressing a CD19 -specific CAR were pre -incubated with
either a control antibody or an anti-CD28 antibody, then co-cultured with Calcein-AM–labeled
721.221 cells at an effector -to-target (E:T) ratio of 20:1. The experiment was repeated two
times and performed in six replicates. *** P < 0.001, Student’s t-test.
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Supplementary Figure 6. Safety of irradiated YTS-CAR cells. (A,B) YTS cells expressing
a CD19-targeted CAR were either left non -irradiated or irradiated at the indicated doses and
subsequently assessed for viability and metabolic activity. (A) Viable cells were quantified by
flow cytometry for up to 30 days. (B) Me tabolic activity was evaluated using an MTT assay
for up to 15 days. Follow-up of non-irradiated cells was limited to day 7, as the culture medium
was not replenished. (C) Irradiated and frozen YTS-CAR cell numbers were monitored up to
20 days by flow cytometry using viability gating. Fresh non -irradiated cells were used as
control. (D) Cytotoxic activity of YTS -CAR CD19 cells over time. The cytotoxic activity of
frozen-thawed, irradiated or non-irradiated YTS FLAG-CAR CD19 cells was assessed against
Raji target cells at different time points (E:T ratio = 10:1) using a flow cytometry–based killing
assay. The experiment was repeated twice. A complementary cytotoxicity assay using Calcein-
AM labeling was also performed and showed similar trends. (E) In vivo persistence of
irradiated YTS-CAR CD19 cells. 20X106 irradiated and frozen YTS-CAR CD19 cells were
injected into NSG mice, and their presence in peripheral blood was analyzed by flow cytometry
at different time points after infusion. Data are shown for five individual mice at the indicated
time points. (F) EBV transfer assessment: HEK293T cells were co -cultured with YTS -CAR
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CD19 cells at an effector -to-target ratio of 5:1 for 24 h, followed by removal of YTS cells.
After 5 days, genomic DNA was extracted from HEK293T cells and EBV presence was
assessed by qPCR targeting BALF5.
Supplementary Figure 7. Comparison of YTS -CAR and NK -92 CAR proliferation and
representative tumor images from an in vivo experiment. (A) Comparison of proliferation
of YTS-CAR and NK-92 CAR cells. YTS -CAR and NK-92 CAR cells were seeded at equal
densities ( 0.5X106 cells/mL) and monitored over time by flow cytometry using viability
staining. Data shown are from one representative experiment out of two performed, each
conducted in triplicate. (B) Representative images of tumors collected from the experiment
shown in Figure 5B.