PMCID string | Title string | Sentences string |
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PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Next, we evaluated the efficacy of 15–4P-stimulated ex vivo activated allogeneic B6 NK cells (H-2b) against Neuro2a (H-2a) murine NBL to evaluate the impact of Ly49 mismatch on cytotoxicity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Neuro2a NBL targets were co-cultured with either IL-15/IL-15Rα expanded allogeneic NK cells or 15–4P expanded allogeneic NK cells for 24 hours. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Apoptotic Neuro2a cells were selected by size and fluorescence intensity of caspase 3/7 and quantified using live-cell imaging by the IncuCyte system. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | 15–4P-expanded allogeneic NK cells exhibited slightly increased target killing compared with IL-15-expanded allogeneic NK cells, despite showing a significant decrease in Fas-L expression (figure 2B). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Surface TIM-3 upregulation was confirmed on IL-15/IL-15Rα NK cells and 15–4P stimulated NK cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Results showed a significantly higher percentage of TIM-3 cells (20.78%±1.695) (p=0.0006) and TIM-3 expression by MFI in 15–4P stimulated NK cells relative to IL-15/IL-15Rα NK cells (4.134%±0.1592) (p=0.0012) (figure 2D, E). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | The elevated expression of TIM-3 on AgN2a-4P NK cells combined with elevated expression of putative TIM-3 ligands on NBL cells suggests this immune checkpoint may be a relevant axis governing NK cell function. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Initially, we measured IFN-γ and tumor necrosis factor (TNF)-α mRNA levels to determine the impact of TIM-3 blockade on NK cell cytokine production after ex vivo expansion. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | We co-cultured IL-15 B6 allogeneic NK cells or 15–4P stimulated B6 allogeneic NK cells with isotype control or anti-TIM-3 antibody in the presence of Neuro2a NBL cells at an effector-to-target (E:T) ratio of 10:1 and assessed cytokine production. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | TNF-α production was significantly increased in the 15–4P NK cells compared with IL-15 NK cells; however, there was no increase after 15–4P NK cell with TIM-3 blockade after exposure to Neuro2a NBL. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Similarly, we observed a notable increase in IFN-γ production in 15–4P stimulated NK cells compared with IL-15 NK cells, but 15–4P NK cells showed no further increase in IFN-γ (online supplemental figure 2D). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Then, to investigate whether blocking TIM-3 could enhance the killing potential of 15–4P stimulated NK cells, we co-cultured 15–4P stimulated NK cells with Neuro2a at an E:T ratio of 10:1 with isotype control or anti-TIM-3 and then assessed surface expression of activating receptors and death ligands as well as cytotoxicity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | There was a high percentage of NKG2D and NKp46 cells and elevated surface expression of NKG2D and NKp46 by MFI among 15–4P stimulated NK cells as seen in prior experiments, but these subsets were not enriched by TIM-3 blockade, suggesting NK cells were already maximally activated. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | After exposure to Neuro2a, 15–4P NK cells with TIM-3 blockade exhibited a higher percentage (p=0.004) but not MFI (p=0.05) of TRAIL than without the anti-TIM-3 antibody. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Compared with 15–4 P cells alone, TIM-3 blockade enhanced both the percentage (p=0.02) of Fas-L 15–4P NK cells, and Fas-L MFI (p=0.007) compared with 15–4P NK cells after Neuro2a exposure (figure 3A). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | This experiment was repeated with NXS2 NBL cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Interestingly, 15–4P NK cells treated with TIM-3 blockade showed no difference in NKG2D and NKp46 expression by MFI as compared with 15–4P NK cells (online supplemental figure 3A). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Additionally, in the presence of NXS2, we found no significant increase in TRAIL, Fas-L, granzyme B or perforin per cent or expression, suggesting that blockade of exposure to TIM-3 ligands on NBL is required to augment allogeneic NK cell activation (online supplemental figure 3B). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Next, we evaluated cytotoxicity by quantifying CD107a degranulation on allogeneic NK cells following exposure to NBL. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Interestingly, we found no discernible difference in degranulation between 15–4P stimulated NK cells with or without TIM-3 blockade in response to NXS2, which have lower expression of TIM-3 ligands (online supplemental figure 3C). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, TIM-3 blockade resulted in a significant increase in the percentage of CD107a+15–4P NK cells, as well as MFI of CD107a granules (figure 3B), in response to Neuro-2a. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Additionally, we observed a significant increase in the percentage of granzyme B-positive cells following TIM-3 blockade, as well as a significant increase in both granzyme B and perforin expression (figure 3C). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | To determine the impact of TIM-3 blockade on NK cell maturation, we assessed the expression of the transcription factors T-box expressed in T cells (T-bet) and Eomesodermin (Eomes) after exposure to NBL tumor cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | TIM-3 blockade did not impact T-bet per cent or expression as 15–4P NK, and 15–4P NK with TIM-3 blockade, cells showed similarly low levels of T-bet. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, we observed a significant rise in both the percentage (p=0.004) and MFI (p=0.004) of Eomes in 15–4P stimulated NK cells after exposure to TIM-3 blockade (47.05±1.607) compared with 15–4P NK cells alone (38.33±0.6756) (p=0.0018). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | This was consistent with Eomes MFI (figure 3D). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | We determined the ratio of Eomes positive cells relative to T-bet in 15–4P stimulated NK cells and 15–4P stimulated NK cells with anti-TIM-3 after Neuro2a NBL exposure; however, there was no significant difference between the two groups (figure 3E). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Interestingly, TIM-3 blockade did not enhance Eomes expression in 15–4P NK cells after exposure to NXS2 NBL cells which have lower expression of TIM-3 ligands (online supplemental figure 3D). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | These findings suggest that TIM-3 blockade enriches for NK cells in early maturation. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Following treatment with anti-TIM-3, 15–4P stimulated NK cells exhibited heightened expression of death ligands and increased markers of degranulation, indicating a phenotype conducive to antitumor activity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Allogeneic NK cells were incubated with Neuro2a NBL with anti-TIM-3 or an isotype control, and apoptotic NBL cells were quantified using IncuCyte live cell imaging of caspase 3/7 expression. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | For all groups, peak killing occurred after the 8-hour mark. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | 15–4P NK cells treated with anti-TIM-3 antibody exhibited significantly higher killing compared with all other groups (figure 3F). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | The enhanced killing capacity of 15–4P NK cells was also observed in the presence of a second murine NBL cell line, 9464D (online supplemental figure 3E). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | This effect was not observed against NXS2 (figure 3G). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | We conducted a comparative evaluation of IL-15 allogeneic NK cells and 15–4P expanded allogeneic NK cells following exposure to the NBL TME to investigate whether TIM-3 blockade modulated NK cell gene expression. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Total RNA was isolated from 15 to 4P stimulated NK cells NK cells co-cultured with Neuro2a in the presence of isotype controls or anti-TIM-3 for 4 hours. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Using the NanoString/nCounter Mouse PanCancer Immune Profiling Panel, the expression of 760 genes was examined. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | The comparison between 15–4P stimulated NK cells and 15–4P stimulated NK cells treated with TIM-3 blockade showed 25 significantly upregulated genes, and 12 genes showed significant downregulation compared with 15–4P stimulated NK cells treated with an isotype control. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Within the upregulated subset of genes, numerous genes associated with NK cell trafficking and target cell recognition (CCL4, CCL3, CCL1, XCL1, CXCR6), and the non-canonical NF-κB pathway (LTA, LTB, TNFSF10, TNFSF14, TNFSF8, RELB, TNF, TRAF3) were observed. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Both ICAM1, which encodes CD54, and TNFSF10, which encodes TRAIL, exhibited a fold change of 2.4 (with false discovery rate p values of 0.003 and 0.0024, respectively). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Furthermore, genes encoding IL-15 and IL-21 receptors were upregulated. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Surprisingly, Prf1 (perforin) was downregulated in IL-15/AgN2a-4P/TIM-3 NK compared with 15–4P stimulated NK cells despite TRAIL upregulation (figure 4A, B; online supplemental table 1). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | This alteration may suggest functional impairment due to exhaustion. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Following the initial granule-mediated killing phase, NK cells may switch to death receptor signaling to sustain serial killing on granule depletion. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | NK cells use multiple cytotoxic pathways in tumor surveillance, transitioning between granule release and death receptor-mediated killing to initiate and sustain long-term cytotoxic activity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | To assess the impact of multiple NK cell cytotoxicity pathways on the killing efficiency of 15–4P stimulated NK cells in the presence of TIM-3 blockade, we exposed allogeneic NK cells to Neuro2a NBL in the presence of blocking antibodies targeting NKG2D, TRAIL, and Fas-L. The inhibition of all three targets resulted in a significant reduction in NK cell killing. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Notably, the blockade of Fas-L completely ameliorated NK cell cytotoxicity against NBL compared with NKG2D or TRAIL blockade, suggesting there is a hierarchy of pathways used to mediate NK cytotoxicity of NBL when TIM-3 is engaged (figure 4C). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | These data underscore the potential of TIM-3 blockade to enhance NK cell cytotoxicity against NBL, particularly in tumors with high expression of TIM-3 ligands, emphasizing the importance of considering the makeup of the NBL TME in therapeutic strategies. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Given our observations regarding the positive impact of TIM-3 blockade on the phenotype and function of 15–4P stimulated NK cells in vitro, we hypothesized that this therapy could yield therapeutic benefits in vivo. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, the intricate pro-inflammatory environment post-allo-HCT and the immunosuppressive NBL TME present additional challenges that could impede NK cell cytotoxic efficacy. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | To address this concern, we evaluated the potency of the combination allogeneic NK cell treatment against Neuro2a NBL following allo-HCT. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Initially, we established an MHC-mismatched haploidentical allo-HCT model wherein lethally irradiated B6AJF1 (H-2b × H-2a) recipients received 5×10 T cell-depleted (TCD) B6 (H-2b) bone marrow with 1×10 B6 T-cell add back on day+0 to simulate residual T cells present in the donor graft during clinical T-cell depletion protocols (eg, CD3 or αβ-TCR depletion). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | On day+7, mice were subcutaneously inoculated with Neuro2a (H-2a) cells to mimic relapse post-HCT. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Recipients then were infused with 3 weekly infusions of B6 IL-15 allogeneic NK cells or B6 15–4P stimulated allogeneic NK cells administered with either an isotype control or anti-TIM-3 treatment every 7 days starting on day+9 (figure 5A). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Although TIM-3 blockade has not been tested after haploidentical allo-HCT, despite the presence of residual T cells in the donor bone marrow graft, we did not observe significant differences in GVHD-associated weight loss (figure 5B). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | By day 35, mice treated with 15–4P NK cells and anti-TIM-3 exhibited the most substantial reduction in tumor growth compared with controls (figure 5C). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Treatment with 15–4P allogeneic NK cells combined with TIM-3 blockade significantly prolonged OS compared with mice treated with 15–4P stimulated NK cells and an isotype control, anti-TIM-3 alone, IL-15 NK cells alone, IL-15 NK cells alone with anti-TIM-3, or no treatment groups (figure 5D). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Given the known role of TIM-3 blockade in promoting T-cell responses and the presence of a low dose of T cells in the graft, we sought to delineate the contributions of NK cells and T cells to graft-versus-tumor (GVT) effects and OS in our allo-HCT model by depletion of these cell subsets prior to treatment. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | In our T-cell depletion studies, B6AJF1 mice received T-depleted B6 bone marrow without T-cell add-back. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | In NK depletion studies, B6AJF1 mice received NK-depleted B6 bone marrow with T-cell add-back. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Since NK cells typically are the first lymphocyte to reconstitute after allo-HCT, anti-NK1.1 antibody was administered intraperitoneally every 4 days to maintain NK cell depletion. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | NK1.1 depletion was performed to ensure the successful engraftment of allogeneic NK cells as endogenous NK cells can compete for space within the bone marrow niche and induce rejection of donor NK cells due to alloreactivity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Subsequently, mice were inoculated with Neuro2A cells and treated with anti-TIM-3 antibody alone or B6 15–4P stimulated allogeneic NK cells with anti-TIM-3 antibody (figure 6A). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Consistently, there were no noticeable differences concerning GVHD-associated weight loss (figure 6B). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | NK cell depletion led to a significant decrease in OS, while TCD mice exhibited significantly increased survival like that of mice treated with 15–4P stimulated NK cells and TIM-3 blockade (figure 6C). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Furthermore, NK cell depletion resulted in larger tumors in all mice, with no significant difference compared with the control, whereas T-cell depletion led to a significant decrease in tumor growth (figure 6D). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | These findings suggest that while T cells are present in the graft, NK cells may play a more substantial role in the survival benefit conferred by 15–4P stimulated NK cells, possibly due to the limited number of T cells compared with NK cells resulting from repeated adoptive transfers. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Moreover, these results underscore the crucial role of NK cells as the primary drivers of TIM-3-induced therapeutic benefits against NBL after allo-HCT, given that similar outcomes were not observed with anti-TIM-3 treatment alone (online supplemental figure 4A, B). |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | T cell-depleted (TCD) allo-HCT has recently gained traction as a safe alternative to standard unmanipulated allo-HCT in order to leverage the GVT effect mediated by donor-derived innate effector cells, like NK cells, while mitigating lethal GVHD. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | While graft manipulation techniques like CD3 or αβ-TCR depletion have achieved up to 5 log10 depletion of T cells, there are always depletion-resistant CD8+ or CD4+ T cells to consider toward impacting GVT or GVHD. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | A major limitation of TCD allo-HCT is the increased risk of infectious complications and relapse due to delayed immune reconstitution of T cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Preclinically, we have previously explored the use of TCD haploidentical allo-HCT in combination with adoptively transferred ex vivo activated NK cells and an anti-GD2 immunocytokine. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | In this study, we found that 15–4P ex vivo expanded NK cells have increased potency against NBL target cells, especially when combined with TIM-3 blockade. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | CD54 and CD137L co-stimulation have never been combined to activate murine NK cells ex vivo. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | While CD54CD137LAgN2 a-4P cells were initially designed as an in vivo vaccine for stimulating T cells, we demonstrate for the first time that combining IL-15/IL-15Ra with AgN2a-4P exposure ex vivo significantly boosts NK cell proliferation and activation, allowing for adoptive transfer of highly potent immune effector cells in the allo-HCT setting. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | AgN2a-4P stimulation increased both NKG2D and TRAIL expression on NK cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | 15–4P-stimulated NK cells exhibited increased IFN-γ production compared with IL-15/IL-15RA stimulation alone. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Prior studies done in a syngeneic model by Jing et al demonstrated that AgN2a-4P vaccination enacted an anti-NBL response through CD8 and CD4 T cells mediated response, despite their tumor target being the parental line AgN2a, which does not express MHC class II. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | While studies have shown that CD4 T cells can kill tumor targets through an indirect mechanism involving IFN-γ directed elimination, the contribution of NK cells to the anti-NBL response cannot be ruled out. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | NK cell reconstitution occurs rapidly following HCT, and it is possible that AgN2a-4P vaccination induced an NK cell-mediated antitumor response and increased IFN-γ production, which indirectly facilitated CD8 T cell and macrophage antitumor responses. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Additionally, we found that 15–4P stimulation significantly enhanced NK cell-mediated lysis of Neuro2a NBL tumor compared with IL-15 NK cells in vitro. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | IL-15 exposure results in an upregulation of TIM-3 expression, where TIM-3 can act as a negative regulator of activation to limit autoreactivity in innate immune cells and maintain self-tolerance. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Although increased TIM-3 upregulation is associated with activated NK cells, cytotoxicity is significantly impaired on crosslinking of TIM-3 with cognate ligands. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | In addition to TIM-3 regulation of NK cell activation, inhibitory signals in the NBL TME can dampen the NK cell response through mechanisms including blockade of target cell adhesion and inhibition of LFA-1 activation, subjugating a weaker activating stimulus and tilting the NK cell response towards inactivation. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | TIM-3 ligands such as Gal-9, CEACAM-1, HMGB1, and PtdSer are expressed at different levels on and within NBL cells and are present in the TME as they are ancillary to pathogenesis.3436 Our results showed that multiple TIM-3 ligands were expressed at a higher proportion on Neuro2a, but not NXS2, suggesting that TIM-3 ligand exposure is cell line dependent and may only be upregulated in a subset of patients with NBL. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, exposure to inhibitory signals from TIM-3 ligands can originate from tumor-associated macrophages as well as other immune cell types within the TME. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | To better understand the mechanisms driving 15–4P NK cells treated with TIM-3 blockade against NBL, we analyzed upregulation of 25 genes and downregulation of 12 genes in 15–4P stimulated NK cells compared with 15–4P stimulated NK cells alone, including ICAM1 and TNFSF10 encoding CD54 and TRAIL, respectively. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Within the upregulated subset of genes, numerous genes were found to be associated with NK cell trafficking and target cell recognition (CCL4, CCL3, CCL1, XCL1, CXCR6) in 15–4P stimulated NK cells with TIM-3 blockade compared to 15–4P NK cells alone. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Modulating chemokine receptors may contribute to increased NK cell motility, thereby allowing 15–4P NK cells enhanced ability to track Neuro2a NBL cells in the absence of TIM-3 inhibition. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Interestingly, we saw increased expression of multiple genes involved in the TNFR2 non-canonical NF-kB pathway (LTA, LTB, TNFSF10, TNFSF14, TNFSF8, RELB, TNF, TRAF3) and in DAP12 signaling (H2-Aa, KLRC2), which suggests the involvement of NKG2D activation in anti-TIM-3 15–4P NK cell mediated killing. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Functional cytotoxicity studies confirmed the ability of 15–4P NK cells treated with TIM-3 blockade to kill Neuro2A targets, which were reversed when blocking NKG2D, TRAIL, or Fas-L. Interestingly, we observed a downregulation of Prf1 (perforin) in 15–4P NK cells treated with TIM-3 blockade compared with 15–4P stimulated NK cells, while TNFSF10 (TRAIL) showed upregulation. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | This may suggest functional impairment in 15–4P NK cells treated with TIM-3 blockade, potentially due to exhaustion. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | The upregulation of TRAIL and the non-canonical Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway intermediates suggests a compensatory mechanism, where NK cells engage the death receptor pathway for killing on granule depletion, thereby sustaining serial killing of target cells. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, NK cells are regulated by a complex interplay of activating and inhibitory co-receptors which contribute to a dynamic response. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Therefore, it cannot be ruled out that unidentified alternative signaling pathways may influence TIM-3 induced NK cell activation. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Further investigation is required to understand how TIM-3 engagement mobilizes FAS-L and TRAIL in the context of NK cell biology. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | Several studies have indicated that blocking TIM-3 interactions during hepatitis B virus infection, as well as in melanoma, acute myelogenous leukemia and multiple myeloma, resulted in enhanced NK cell cytotoxicity.3942 Conversely, other studies have reported enhanced IFN-γ production without a corresponding improvement in NK cell degranulation, suggesting that TIM-3 engagement can shift the NK cell response between positive and negative signaling events through differential tyrosine phosphorylation of the cytoplasmic tail. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | We observed increased IFN-γ production in 15–4P stimulated NK cells, which indicated that exposure to AgN2a-4P during ex vivo expansion shifts NK cells to a more immature phenotype that is still capable of robust cytotoxicity. |
PMC12699621 | TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant | However, the addition of TIM-3 blockade did not further enhance IFN-γ and TNF-α production in 15–4P NK cells. |
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