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PMC10335267
The Roles of Serum CXCL16 in Circulating Tregs and Gastrointestinal Stromal Tumor Cells [Retraction]
When approached for an explanation, the authors were cooperative but were unable to adequately address the concerns raised and could not provide sufficient supporting information.
PMC10335267
The Roles of Serum CXCL16 in Circulating Tregs and Gastrointestinal Stromal Tumor Cells [Retraction]
As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article.
PMC10335267
The Roles of Serum CXCL16 in Circulating Tregs and Gastrointestinal Stromal Tumor Cells [Retraction]
The authors listed in this publication have been informed.
PMC10335267
The Roles of Serum CXCL16 in Circulating Tregs and Gastrointestinal Stromal Tumor Cells [Retraction]
We have been informed in our decision-making by our editorial policies and the COPE guidelines.
PMC10335267
The Roles of Serum CXCL16 in Circulating Tregs and Gastrointestinal Stromal Tumor Cells [Retraction]
The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as “Retracted”.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in patients with HR-NBL; however, graft-versus-host disease (GVHD) and disease progression have limited clinical application.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Ex vivo stimulated allogeneic natural killer (NK) cells represent an approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Ex vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble interleukin-15 (IL-15) and IL-15 receptor alpha (IL-15Rα) alone or with irradiated CD137L/CD54 aggressive variant of the Neuro-2a murine neuroblastoma cell line (15–4P) at a 1:1 ratio for 10–12 days.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Lethally irradiated B6AJF1 mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15–4P B6 NK cells on days 14, 21, and 28.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
In select experiments, T cell and NK cells were selectively depleted to establish contribution to the GVT effect.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
All groups were analyzed for tumor growth, GVHD and survival.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Co-culturing NK cells with 15–4P results in 78-fold expansion with increased expression of Kiel-67 (Ki-67) and Natural Killer Group 2, Member D (NKG2D), NKp46, TNF-Related Apoptosis-Inducing Ligand (TRAIL) and TIM-3.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
15–4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared with IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared with NXS2.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15–4P NK cell degranulation, Eomesodermin, TRAIL and Fas Ligand expression observed.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
In vivo, the combination of 15–4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared with NK cells or anti-TIM-3 alone.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Depletion of NK cells, but not T cells, abrogated the GVT effect.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Allo-HCT can be a platform for treating NBL using combination ex vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.T cell-depleted (TCD) allogeneic hematopoietic cells transplant (allo-HCT) has potential to be a salvage therapy for relapsed/refractory neuroblastoma (NBL) through the graft-versus-tumor (GVT) effect; however, the high incidence of graft-versus-host disease (GVHD) and disease progression has hindered widespread clinical application.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Allogeneic natural killer (NK) cell therapy can be a safe and feasible treatment but has had limited efficacy in NBL.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) blockade has shown encouraging results for a variety of tumors but has not been explored for NBL nor used to enhance the GVT effect.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Ex vivo stimulated allogeneic NK cells demonstrate robust expansion, proliferation, activation and cytotoxicity against NBL which is further augmented by the addition of TIM-3 checkpoint blockade.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
In an allo-HCT model of NBL relapse, mice treated with interleukin-15/IL-15 receptor alpha (IL-15/IL-15Rα) aggressive variant of the Neuro-2a murine neuroblastoma cell line (15–4P) stimulated NK cells and anti-TIM-3 exhibited prolonged overall survival and reduced tumor growth without exacerbating GVHD.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Relapse of NBL after TCD allo-HCT can be successfully treated with the combination of adoptively transferred 15–4P stimulated allogeneic NK cells and TIM-3 checkpoint blockade, representing a novel therapeutic approach that can be translated to the clinic or used as a platform to treat other solid tumors with high TIM-3 ligand expression.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
High-risk neuroblastoma (NBL) is an aggressive extracranial solid tumor of neural crest origin that commonly occurs in children typically under 5 years old.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
About 40% of patients with NBL are considered high risk.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Unfavorable molecular characteristics indicate poor prognosis, requiring intensive treatment regimens including chemotherapy, surgery, consolidation with autologous hematopoietic cell transplant (auto-HCT), radiation therapy and post-consolidation anti-GD2 immunotherapy with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and cis-retinoic acid.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
The addition of anti-GD2 therapy has increased the 5-year overall survival (OS) of high-risk NBL from 20% to 50%, yet half of patients still relapse.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
There is still a critical need for the development of therapies for NBL with lower toxicities.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Auto-HCT has been standard of care for high-risk NBL for decades but offers little benefit outside of stem cell rescue.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Three phase III randomized clinical trials have reported that supratherapeutic chemotherapy with auto-HCT support improved event-free survival (EFS) but failed to ameliorate 5-year OS, which highlights a critical need for incorporating novel therapies for the treatment of high-risk NBL.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Allogeneic HCT (allo-HCT) offers a unique advantage to auto-HCT due to donor-derived lymphocytes.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
However, it has been difficult to translate these results to solid tumors due to the high incidence of graft-versus-host disease (GVHD) and disease progression.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Advances in graft engineering now allow for precise removal of allogeneic T cells (eg, αβ-T cell receptor/TCR depletion, CD3 depletion) that can greatly reduce the risk of GVHD; however, these methods increase the likelihood of relapse, infection, and delays in immune reconstitution.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Adoptive cell transfer of natural killer (NK) cells after T cell-depleted allo-HCT has the potential to improve outcomes by treating NBL relapse while controlling viral infections without inducing GVHD.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells are innate lymphoid cells that play an important role in antitumor surveillance by identifying infected, stressed, or tumorigenic cells through a balance of activating and inhibitory cell surface receptors.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells are not limited by antigen recognition; instead, missing or mismatched binding between killer-cell-immunoglobulin and “non-self” major histocompatibility complex (MHC) class I (or human leukocyte antigens class I) triggers a cytotoxic response.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells do not induce GVHD and can exert a protective defense against GVHD by suppressing allo-reactive T cells.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Clinical studies in NBL have confirmed the safety profile of allogeneic NK cell therapy; however, response rates remain modest.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Due to the biological limitations of naïve NK cells, methods of ex vivo expansion and activation that enhance NK cell cytotoxicity without inducing exhaustion are critical.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Cytokine stimulation with multiple cytokines, including interleukin (IL)-15, has been shown to induce T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) expression on NK cells, suggesting that TIM-3 expression marks a mature and highly activated subpopulation.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
TIM-3 expression increases following cytokine stimulation and promotes interferon (IFN)-γ production, and NK cells with high TIM-3 expression are highly responsive.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
However, cross-linking of TIM-3 with agonist antibodies or cognate ligands significantly inhibits NK cell-mediated cytotoxicity induced by CD16 and NK cell activation and dampens degranulation, making it an ideal target for immune checkpoint inhibition.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Here, we explore the use of an aggressive variant of the Neuro-2a murine neuroblastoma cell line (AgN2a-4P), a murine neuroblastoma cell line engineered from an AgN2a to express four immune costimulatory proteins CD54, CD80, CD86, and CD137L (4P) on its surface.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Notably, CD137L (41BBL) serves as a well-established NK cell activator, driving proliferation and heightened antibody-dependent cellular cytotoxicity via Fc receptor signaling.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Additionally, CD54 can bind to LFA-1 and MAC-1 on NK cells and augments cytotoxicity by promoting adhesion and granule polarization.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
The current investigation explores the impact of adoptive cell therapy of ex vivo AgN2a-4P-stimulated allogeneic NK cells with TIM-3 immune checkpoint blockade for the first time after allo-HCT for NBL as a novel combination immunotherapy.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Female C57BL/6NCr (B6, H-2b), and B6AJF1 (B6AJ, H-2b × H-2a) mice were purchased from the National Cancer Institute Animal Production Program and Charles River Laboratories International (Frederick, Maryland, USA).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Mice were housed in accordance with the Guide for Care and Use for Laboratory Mice and used between 8 and 16 weeks of age at time of experimentation.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Murine bone marrow cells (BMCs) were harvested from tibias and fibulas and processed into a single-cell suspension.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Splenocytes were processed into a single-cell suspension.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
BMCs and splenocytes were pooled.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Red blood cells were lysed with ACK lysis buffer (Lonza, Walkersville, Maryland, USA).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells were isolated using the NK cell isolation kit (Miltenyi Biotec, Auburn, California, USA) by negative selection on the AutoMACs Pro (Miltenyi Biotec).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells were expanded and activated in the presence or absence of irradiated (100 Gy) AgN2a-4P at a 1:1 ratio and cultured in Roswell Park Memorial Institute (RPMI) medium (Corning Life Sciences, Durham, North Carolina, USA) containing 10 ng/mL IL-15/IL-15 receptor alpha (IL-15Rα) conjugate, comprised of recombinant mouse IL-15 protein (R&D Systems, Minneapolis, Minnesota, USA) and recombinant mouse IL-15Rα Fc chimera protein (R&D Systems), 10% fetal bovine serum (GeminiBio, Sacramento, California, USA) with 50 IU/mL penicillin/streptomycin (Lonza), 0.11 mM 2-beta-mercaptoethanol (Gibco, Carlsbad, California, USA), 1× MEM non-essential amino acids (Corning), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Corning), and 1 mM sodium pyruvate (Corning).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NK cells were expanded for 12 days and were supplemented with additional 10 ng/mL IL-15/IL-15Rα and 5 mL of media every 2 days and maintained at 37°C in 5% CO2.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
BMCs were harvested from B6 mice and processed into single-cell suspensions.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
T cells were depleted using a CD3 MicroBead Kit (Miltenyi Biotec) by negative selection on the AutoMACs Pro Separator (Miltenyi Biotec) at day+0.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Separately, T cells were isolated using a pan T Cell Isolation Kit (Miltenyi Biotec) on single-cell suspensions obtained from splenocytes isolated from B6 mice.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
B6AJF1 recipient mice received a single dose of 11 Gy total body irradiation using an X-Rad 320 (Precision X-ray Irradiation, Madison, Connecticut, USA).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Irradiated recipients received retroorbital injection of 5×10 donor-derived BMCs and 1×10 T cells in serum-free RPMI (Invitrogen, Carlsbad, California, USA).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
At day +7, 2×10 Neuro2A NBL cells (H-2a) were prepared as a single cell suspension in serum-free RPMI and injected into B6AJF1 recipient mice subcutaneously on the right hind limb flank to mimic post-HCT relapse.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Tumor length and width were measured biweekly using a digital caliper, and tumor growth was calculated as V=½ (length×width).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Statistics were performed using GraphPad Prism V.9.0 (GraphPad Software, San Diego, California, USA).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Survival curves were plotted using Kaplan-Meier estimates, and the Mantel-Cox log-rank test was used to analyze survival data.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
The two-tailed Mann-Whitney U test (non-parametric data sets) or unpaired t-test (parametric data sets) with Welch’s corrections was used for the statistical analysis when comparing two groups.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
When comparing three or more groups, the Kruskal-Wallis (non-parametric data sets) with Dunn’s multiple comparison post hoc test or one-way analysis of variance (parametric data sets) with Tukey post hoc test.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
P value<0.05 was considered statistically significant.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Refer to online supplemental methods for more information.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
AgN2a-4P is a genetically engineered murine NBL cell line that expresses CD54, CD80, CD86, and CD137L co-stimulatory molecules but has not been tested on allogeneic NK cells previously.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
AgN2a-4P can interact with NK cells through CD137L, binding CD137, and CD54, binding LFA-1/MAC-1 on NK cells (figure 1A).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
To study the impact of AgN2a-4P exposure on NK cell expansion and activation, NK cells from B6 mice were expanded with AgN2a-4P cells with IL-15/IL-15Rα at a 1:1 ratio and assessed by flow cytometry at day 12 (figure 1B).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
B6 NK cells expanded with IL-15/IL-15Rα and AgN2a-4P (15–4P) exhibited a significant increase in the percentage of Natural Killer Group 2, Member D positive (NKG2D) cells (29.27±1.894) compared with IL-15 NK cells (20.80±0.7095).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
While 15–4P did not impact the percentage of NKp46 NK cells (35.08±1.427) in comparison to IL-15 NK cells (33.70±0.3342), the expression of NKp46 by median fluorescence intensity (MFI) increased (p=0.0286) (figure 1C).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
CD80 and CD86 both bind to CD28 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4), which have opposing functions as a stimulatory receptor and inhibitory receptor, respectively.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Primary resting human NK cells have been shown to express a CD28 homolog which activates strong lysis against VISTA/B7-H5 targets on synergy with NKp46 and 2B4.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Cytokine stimulation induces expression of both CTLA-4 and CD28 in murine NK cells which can activate or inhibit IFN-γ release on ligand binding.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
To determine the roles of CD80, CD86, CD54, and CD137L in enhancing 15–4P stimulated NK cell responses, NK cells were expanded in the presence of 15–4P in combination with isotype control, anti-CD54 alone, anti-CD137L alone, and anti-CD80/CD86/CD54/CD137L.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
When cultured with antibodies against all four receptors, there was a significant reduction in the percentage of NKG2D and NKp46 NK cells compared with isotype control.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
When cultured with antibody against CD137L, there was no significant reduction in the percentage of NKG2D and NKp46 NK cells compared with isotype control.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
When cultured with antibody against CD54, there was a significant reduction in NKp46 NK cells, but not NKG2D NK cells compared with isotype control.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
This data supports the importance of NK cell exposure to the co-stimulatory molecule CD54 in the enhanced activation of NK cells (online supplemental figure 1).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Due to the expression of 41BBL on AgN2a-4P, we confirmed whether AgN2a-4P exposure during expansion impacts NK cell proliferation.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
We observed a 78-fold expansion in 15–4P (78.33±20.46) compared with IL-15 NK cells (27.39±1.207) (figure 1D).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
There was a significant increase (p=0.0006) of Kiel-67 (Ki-67) 15–4P NK cells (52.24±3.953) and Ki-67 expression by MFI compared with IL-15 NK cells (13.42±0.318) (figure 1E, F).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
To examine effector capacity, expansion with 15–4P resulted in a significantly higher percentage of TNF-Related Apoptosis-Inducing Ligand (TRAIL) NK cells (19.06±0.6823) compared with IL-15 NK cells (14.10±0.6532) (p=0.0006), and an increase in TRAIL expression by MFI (p=0.0238).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Conversely, 15–4P stimulated NK cells exhibited a significant decrease in Fas Ligand (Fas-L) cells (3.998±0.2588) in contrast to IL-15 NK cells (5.935±0.6996), and there was no difference in Fas-L expression by MFI (figure 1G).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
These findings indicate that exposure to AgN2a-4P during IL-15/IL-15Rα-based NK cell expansion results in comparable activation as NK cells are likely to be maximally activated through expansion with IL-15.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Increased proliferation markers were observed in 15–4P stimulated NK cells compared with NK cells expanded with IL-15/IL-15Rα alone.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Additionally, the potential impact of cytotoxicity depending on if TRAIL versus Fas-L pathways are used to trigger apoptosis of NBL tumors requires further investigation.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
The NBL tumor microenvironment (TME) is highly immunosuppressive, influencing NK cells through a complex interplay of activating and inhibitory receptor signaling.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
TIM-3, a marker of maturation in NK cells, interacts with its cognate ligands, including Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1), Galectin-9 (Gal-9), High Mobility Group Box 1 (HMGB1), and Phosphatidylserine (PtdSer) to exert inhibitory regulation in NK cells.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
We verified the surface expression of MHC I and galectin-9 and CEACAM1 on murine NBL cell lines Neuro2a and NXS2.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Neuro2a displayed a significantly higher percentage of CEACAM1 cells (20.08%±0.9016) (p=0.002), and Gal-9 cells (95.10%±0.4235) (p=0.002) compared with NXS2 (figure 2A).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Both Neuro2a and NXS2 had low expression of MHC-I. The MFI of CEACAM1 (p=0.0047) and galectin-9 (p=0.0049) was also notably higher on Neuro2a cells compared with NXS2 (figure 2A).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Due to their intracellular expression, HMGB-1 and PtdSer messenger RNA (mRNA) were measured.
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Neuro2a showed significantly higher mRNA expression of both HMGB-1 and PtdSer compared with NXS2 (online supplemental figure 2A).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
NXS2 lost expression of CEACAM1, Gal-9, and HMGB-1 when implanted in a mouse flank (online supplemental figure 2B).
PMC12699621
TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant
Neuro2A retained expression of CEACAM1, Gal-9, and HMGB-1 when implanted in a mouse flank (online supplemental figure 2C).