Search Results (337)

Background/Objectives: Chronic pain is a significant clinical challenge, in part due to the absence of reliable objective biomarkers for its evaluation and treatment. Growing evidence indicates that immune cells, including natural killer (NK) cells, are involved in the regulation of pain processes. NK cells are innate cytotoxic lymphocytes whose functional status may mirror underlying pathological pain states. In this study, we investigated μ-opioid receptor (MOR) expression and functional alterations of NK cells in a murine model of neuropathic pain induced by chronic constriction injury (CCI). Methods: Mice were divided into three groups: Sham (sciatic nerve exposure without ligation), CCI 14-day, and CCI 21-day groups. At the respective time points, animals were sacrificed and spleens were collected for analysis. Splenocytes were isolated by mechanical dissociation followed by centrifugation and erythrocyte lysis. Lymphocytes were analyzed by flow cytometry to evaluate MOR expression in NK cells and their degranulation activity (CD107a assay). Cells were incubated with fluorochrome-conjugated antibodies against NK cell markers (NK1.1, CD3, Ly49A, Ly49C/I) in combination with anti-MOR and anti-Interferon γ antibody (IFN-γ). Immunofluorescence and confocal microscopy analyses were performed to assess MOR localization and granzyme localization, supporting CD107a-mediated degranulation. Results: Flow cytometry analysis revealed a significant reduction in surface MOR expression on total NK cells from CCI mice compared with sham controls at 14 and 21 days post-injury, a finding corroborated by immunofluorescence evidence of MOR cellular internalization. Functionally, CCI induced a marked decrease in CD107a expression and impaired IFN-γ production both under basal conditions and following PMA/ionomycin stimulation, indicating a hyporesponsive state of NK cells. Consistently, confocal microscopy revealed extracellular release of Granzyme A following CCI, suggesting dysregulated degranulation. Conclusions: Neuropathic pain is associated with a remodeling of NK cell phenotype and effector functions, characterized by impaired cytotoxic activity and cytokine production, along with modulation of inhibitory receptor expression. Notably, MOR-reduced surface expression in NK cells emerges as a potential biomarker of neuropathic pain. Further studies are needed to elucidate the molecular mechanisms regulating MOR expression and its relationship with NK cell hyporesponsiveness and degranulation in chronic pain conditions. Full article

Background: Bronchoscopic cryotherapy may enhance anti-tumor immunity and improve the effect of immune checkpoint blockade, but CD8+ T cell dynamics after cryotherapy combined with pembrolizumab-based therapy in metastatic non-small cell lung cancer (NSCLC) remain insufficiently characterized. Methods: In this prospective, exploratory, randomized, controlled, single-center study, metastatic NSCLC patients were assigned to bronchoscopic cryotherapy performed 7 ± 1 days before first-line pembrolizumab-based therapy or to standard treatment alone. Peripheral blood mononuclear cells were analyzed by flow cytometry at baseline, week 3, and week 6. CD8+ T cell subsets defined by CD45RO, CD28, granzyme B (GzB), IFNγ, Ki-67, and PD-1 were evaluated in relation to treatment group, radiologic response, progression-free survival (PFS), and overall survival (OS). Results: Flow cytometry was performed in 76 patients, including 34 in the cryotherapy group and 42 in the control group. Cryotherapy was associated with a treatment-specific increase in circulating GzB+ CD8+ T cells by week 6. In contrast, Ki-67+ CD8+ and GzB+Ki-67+ cells increased in both treatment groups, suggesting that early peripheral CD8+ proliferation was largely shared across pembrolizumab-based therapy. Radiologic responders demonstrated more sustained proliferative CD8+ dynamics, most consistently reflected by increased CD28+ Ki-67+ cells. In exploratory landmark survival analyses, a higher week-3-to-baseline GzB+ Ki-67+ CD8+ ratio showed a hypothesis-generating association with longer PFS and OS. Conclusions: In metastatic NSCLC patients receiving first-line pembrolizumab-based therapy, the addition of bronchoscopic cryotherapy was associated with peripheral CD8+ T cell remodeling toward enhanced cytotoxic activity. Proliferative CD8+ T cell changes were associated with radiologic response and better survival. These findings support bronchoscopic cryotherapy as a potential immune-modulating adjunct and warrant validation in larger studies. Full article

Synthetic polyinosinic:polycytidylic acid (poly(I:C)) offers an attractive cancer therapeutic by operating on two fronts at once, combining direct tumor cell killing with immunostimulatory activity. Yet, these dual functions can only be efficiently harnessed when intracellular delivery is sufficiently effective to enable poly(I:C) to reach and activate its intracellular receptors. We addressed this delivery challenge by developing pH-responsive formulations using lipoamino fatty acid xenopeptide (LAF-XP) carriers, composed of polar cationizable succinoyl tetraethylene pentamine (Stp) and apolar cationizable LAF building blocks in defined architectures. In particular, poly(I:C)-lipid nanoparticles (LNPs) formulated with bundle LAF₄-Stp₁ XP carriers displayed increased anti-tumoral activity at decreased dosage across multiple cancer cell models, compared to control formulations. In parallel, LAF-XP LNP-delivered poly(I:C) activated immune responses, including CXCL10 production by tumor cells, and activation of peripheral blood mononuclear cells (PBMCs), characterized by increased phenotypic markers (CD69 and LAMP-1/CD107a) and functional molecules (e.g., IFN-γ and granzyme B). Conditioned supernatant of pre-stimulated PBMCs with poly(I:C) reduced cancer cell viability, highlighting the contribution of PBMC-released factors to cancer cell death. Of particular novelty is the combination of poly(I:C) with siRNA-mediated survivin knockdown to increase apoptosis in cancer cells using the bundle LAF-XP LNP. Collectively, our findings establish efficient LAF-XP LNPs as a versatile platform that supports multi-layered therapeutic strategies. Full article

Breast cancer is a type of cancer with the highest incidence and mortality rates among women. PD-L1 suppresses the proliferation and activation of T cells, thereby enabling cancer cells to evade immune surveillance and facilitating tumor progression. Micheliolide (MCL) is a guaianolide-type sesquiterpene lactone with broad biological activities. Our results revealed that radiation upregulates PD-L1 expression in breast cancer cells, while MCL pretreatment can inhibit this effect. Bioinformatics analysis combined with shRNA interference experiments confirmed that radiation upregulates PD-L1 by activating the IRF1-STAT1 signaling pathway, while MCL represses PD-L1 transcription by suppressing this pathway. In addition, MCL also downregulates PD-L1 protein level through accelerating proteasomal degradation of PD-L1. In vivo experiments demonstrated that MCL combined with radiotherapy significantly inhibits the growth of syngeneic tumors and increases intratumoral CD8⁺ T cell infiltration and the frequencies of granzyme B-positive cells. Taken together, our results indicate that MCL enhances T-cell-mediated antitumor immunity and improves radiotherapy efficacy through inhibiting IRF1-STAT1 signaling pathway-driven PD-L1 transcription and promoting PD-L1 protein degradation. This study provides a theoretical basis for the clinical application of MCL as an immunomodulator and radiosensitizer. Full article

The evaluation of therapeutic response is essential in disease monitoring both for disease status and treatment efficacy in inflammatory bowel disease. Here, we focused on the use of positron emission tomography directed towards granzyme B, a serine protease released by activated cytotoxic T cells and natural killer cells, to evaluate the dynamics of therapeutic response in a colitis model. The goal was to explore the use of granzyme B positron emission tomography as a non-invasive biomarker to monitor disease activity and therapeutic response across several treatments in a dextran sulfate sodium-induced colitis model. C57BL/6 interleukin-10 knockout mice were divided into five groups, including a negative control, positive control and three treatment arms (antitumor necrosis factor, prednisolone, and anti-interleukin-23). The negative control group received regular water, while all other groups were induced with colitis via 3% DSS water for 1 week followed by normal water. Treatments were initiated after colitis was induced (anti-TNF antibody, prednisolone, or anti-IL-23 antibody). Positron emission tomography/computed tomography imaging with 68Ga-NOTA-GZP was performed at baseline (after colitis induction, before therapy), and at 1 and 2 weeks after treatment initiation. Histological analyses were also performed at 1 and 2 weeks after treatment initiation. Gzmb expression and histological changes were also assessed with immunofluorescence staining and bulk ribonucleic acid sequencing. Gzmb-targeted PET imaging revealed distinct longitudinal patterns of colonic tracer uptake related to treatment response. In positive control mice with DSS colitis (no treatment), bowel uptake of ⁶⁸Ga-NOTA-GZP increased significantly from baseline to week 2. Anti-TNF treatment reduced granzyme B positron emission tomography uptake significantly at week 2, approaching levels seen in negative controls. In prednisolone-treated mice, ⁶⁸Ga-NOTA-GZP uptake decreased at week 1 but rose significantly by week 2 but still was in normal range. Anti-IL-23 therapy produced a significantly elevated Gzmb PET signal at week 1, followed by a significant decline by week 2 of treatment. The imaging trends were corroborated by tissue analyses and IF staining for Gzmb, which revealed no colonic expression in negative controls and strong Gzmb elevation in positive controls and the prednisolone group but a decreased Gzmb signal in the anti-TNF and late anti-IL-23 groups. Bulk RNA sequencing also supported these findings, with Gzmb gene expression tracking with inflammation severity and NK/T cell abundance and decreasing after effective therapy. Gzmb-targeted PET/CT allows for dynamic and non-invasive assessment of intestinal immune compartment activity and an assessment of therapy in colitis. Gzmb PET was able to detect initial treatment responses of anti-TNF, steroid and anti-IL-23 based on changes in the Gzmb PET signal. This suggests that clinical Gzmb PET imaging may serve as precision imaging for monitoring disease activity with treatment in IBD and help improve patient care by identifying responders and non-responders in real time. Full article

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal microenvironment and profound hypoxia, which contribute to therapeutic resistance. Using an in vitro system incorporating pancreatic cancer cells and cancer-associated fibroblasts (CAFs), we show that hypoxia suppresses CD8⁺ T cell accumulation and, in combination with cancer cell- and CAF-derived factors, further impairs T cell fitness by increasing cell death and reducing proliferation. Although the combination of hypoxia and cancer cell/CAF-derived factors enhances IFNγ and granzyme B expression in CD8⁺ T cells on a per-cell basis, the overall number of functional effector T cells is markedly reduced. Analysis of human PDAC single-cell RNA sequencing data corroborates these findings, revealing that CD8⁺ T cells enriched for hypoxia signatures exhibit elevated apoptosis and stress-response pathways. Furthermore, hypoxia is associated with downregulation of stemness-related genes and upregulation of terminal differentiation markers. Together, these data suggest that the integration of intrinsic hypoxic responses and extrinsic cues from tumor cells and CAFs impairs CD8⁺ T cell fitness and correlates with a terminally differentiated, dysfunctional T cell state. Full article

Vitiligo is characterized by epidermal melanocyte destruction, with autoreactive CD8⁺ T cells playing a central pathogenic role, yet the mechanisms driving their hyperactivation remain unclear. Lactate has emerged as a key immunometabolite that functions as both a signaling molecule and an epigenetic modulator via protein lactylation. Nevertheless, the role of lactate in vitiligo pathogenesis has not been explored. Here, we report that serum lactate levels are significantly elevated in vitiligo patients and correlate positively with disease activity. In a mouse model, lactate administration accelerated vitiligo progression, accompanied by increased CD8⁺ T cell infiltration and melanocyte destruction in lesional skin. In vitro, lactate enhanced CD8⁺ T cell effector molecule expression (granzyme B, perforin, IFN-γ, CD107a) and cytotoxic function. Mechanistically, lactate increased global protein lactylation in CD8⁺ T cells, with marked enrichment at histone H3 lysine 9 (H3K9). H3K9 lactylation (H3K9la) was associated with enhanced chromatin accessibility and transcriptional activation of effector genes, as revealed by RNA sequencing and CUT&Tag analyses. Pharmacological inhibition of lactate production or lactylation abrogated these effects. Collectively, our findings identify lactate as a critical driver of CD8⁺ T cell pathogenicity in vitiligo through H3K9la-mediated epigenetic reprogramming, highlighting lactate metabolism and lactylation as potential therapeutic targets. Full article

Background and Objectives: The incidence of colorectal cancer (CRC) in developed Western countries is constantly growing. CRC represents the third most common cancer and the second leading cancer-related cause of death worldwide. Innate and adaptive immunity play a pivotal role in the tumor response, but many of these interactions are still not well understood. Granulysin (GNLY) is an effector, cytolytic molecule, present in human cytotoxic granules of different lymphocyte subpopulations, mainly in cytotoxic T cells and NK cells. Pore-forming proteins GNLY, perforin and granzymes play a key role in cell-mediated immune responses against tumors and infections. Materials and Methods: We aimed to analyze perforin and GNLY-mediated cytotoxicity in the peripheral blood of patients with CRC by flow cytometry. Simultaneously, the cells were labeled with monoclonal antibodies against perforin, GNLY and different surface antigens (CD3, CD4, CD8 and CD56). Phenotypes of lymphocyte subpopulation and expression of perforin and GNLY were analyzed using intracellular and surface immunofluorescence. Results: Total perforin and GNLY expressions in peripheral blood mononuclear cells (PBMC) were significantly lower than in the control group. Statistically significant differences were observed in the distribution of perforin and GNLY expression in different stages of tumors classified according to Dukes’, indicating that the percentage of total perforin and GNLY was significantly diminished in accordance with tumor progression. Perforin and GNLY expression were significantly reduced in NK and NKT cells, accompanied by reduced cytolytic potential in patients with CRC and a consequent reduction in their ability to eliminate tumors and infected cells. Conclusions: The determination of cytotoxic potential may provide a valuable assessment of a patient’s immune status and represent a novel therapeutic target. Patients with CRC exhibit markedly impaired perforin- and GNLY-mediated cytotoxicity that correlates with disease progression. Assessment and restoration of cytolytic potential may therefore serve as indicators of immune competence and promising therapeutic strategies to improve perioperative and oncologic outcomes. Full article

Background: With the evolving paradigm of vaccine development, microcapsules have attracted considerable research interest as particulate adjuvants over the past decades. However, the rational engineering design of microcapsule-based composite adjuvant systems to elicit robust immune responses remains a significant challenge. Methods: This study developed polylactic acid (PLA) microcapsules with spatiotemporally coupled delivery and immunopotentiator properties. The resulting formulations were assessed for humoral and cellular immune responses in mice. Results: We prepared uniform-sized microcapsules (MC) and formulated them with monophosphoryl lipid A (MPLA) as a composite component (MPLA@MC), with hydrodynamic diameters of 4.58 μm and 4.12 μm, respectively. Such composite adjuvants, when loaded with ovalbumin (OVA) to form OVA@MC and OVA&MPLA@MC, promoted cellular uptake and activation, exhibiting preferred lysosomal escape advantages. For in vivo experiments, microcapsule-based vaccines elevated serum levels of IgG antibody, and OVA&MPLA@MC induced Th1-biased antibody responses. Specifically, OVA&MPLA@MC also elicited strong cellular immune responses compared to other vaccines, as evidenced by increased secretion of Interferon-γ (IFN-γ) in mouse splenocytes and Granzyme B (Gzmb) in T cells. Mechanistically, muscle tissues at the injection site showed that microcapsule-based vaccines enhanced the recruitment for phagocytosis. Meanwhile, bulk RNA sequencing (RNA-seq) confirmed extensive activation of immune responses and related signaling pathways. Conclusions: This rationally designed composite strategy for spatiotemporally coupled delivery serves as a potent platform for orchestrating synergistic immune responses, opening up new avenues for the development of effective therapeutic and anti-infectious vaccines. Full article

Triple-negative breast cancer (TNBC) is characterized by prominent neutrophil infiltration; however, its significance remains controversial. Here, we investigated the role of neutrophil chemoattractant receptors in TNBC progression and metastasis. In contrast to wild-type (WT), Fpr1^−/−, and Fpr2^−/− mice, neutrophils were almost completely absent in 4T1 tumors from Cxcr2^−/− mice, indicating a dominant role for CXCR2 in the recruitment of tumor-associated neutrophils, leading us to use Cxcr2^−/− mice for further studies. Primary tumor growth was comparable between WT and Cxcr2^−/− mice, whereas lung metastasis was significantly increased in Cxcr2^−/− mice, with reduced expression of inflammatory cytokines, chemokines and cytotoxic molecules, including granzyme B and perforin, in primary tumors and metastatic lungs of Cxcr2^−/− mice. In vitro, WT, but not Cxcr2^−/−, neutrophils enhanced CD8⁺ T cell activation, partly via ICAM-1, and directly induced tumor cell death, supporting their anti-tumor function. To assess clinical relevance, transcriptomic data were analyzed. High neutrophil infiltration combined with elevated CXCR2 expression, and to a lesser extent CXCR1 expression, was associated with improved prognosis in patients with basal-like BC that largely overlaps with TNBC. Collectively, these findings suggest that CXCR2-mediated neutrophil recruitment exerts protective, anti-tumor effects and may represent a new prognostic marker for TNBC patients. Full article

Background: B-cell malignancies have been effectively treated using chimeric antigen receptor-T (CAR-T) treatment employing traditional αβT cells. However, because of several obstacles, application in acute myeloid leukemia (AML) is still restricted. A safer “off-the-shelf” alternative can be supplied by CAR-γδT cells, which have major histocompatibility complex (MHC)-independent tumor identification capabilities and a decreased risk of graft versus host disease (GvHD). This study aimed to develop FLT3-targeted CAR-γδT cells that co-express cytokines (IL-2 or IL-7) to increase their anti-AML persistence and therapeutic efficacy. Methods: FLT3-CAR-γδT cells, FLT3-IL2-CAR-γδT cells, and FLT3-IL7-CAR-γδT cells were constructed. Their antitumor potency was comprehensively assessed through cytotoxicity assays, cytokine release, and persistence evaluation in vitro (using AML cell lines and primary AML cells) and in vivo (via mouse model). Results: Superior cytotoxicity against AML cell lines (OCI-AML3, MOLM-13, THP-1, and MV4-11) was demonstrated by FLT3-IL2-CAR-γδT cells, which also released higher levels of granzyme B, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). FLT3-IL2-CAR-γδT cells exhibited cytotoxicity in some primary AML cells in vitro. During the antigen-repeated stimulation assay, FLT3-IL2-CAR-γδT cells preserved the stem cell-like memory T (T_SCM) cell subsets, sustained cytokine release, and maintained excellent viability. FLT3-IL2-CAR-γδT cells considerably slowed the development of AML in vivo and extended the existence (>68 days) of mice. Conclusions: FLT3-IL2-CAR-γδT cells exhibit potent and durable anti-AML activity, providing a novel strategy for clinical AML immunotherapy. Full article

Cancer is a major health concern, with its incidence rate continuing to increase. There is growing interest in the microbiota and its role in carcinogenesis, as it significantly influences physiological and pathological processes. Various aspects of the microbiome have been shown to have both anti-tumor and pro-tumor effects. Advances in techniques such as high-throughput DNA sequencing have greatly improved our understanding of microbial populations in the human and canine gut. We aimed to (1) characterize the intestinal microbiota of healthy dogs and dogs with cutaneous mast cell tumors (MCTs), (2) assess changes in the intestinal microbiota of dogs undergoing electrochemotherapy (ECT) combined with gene electrotransfer (GET) of the IL-12 plasmid (IL-12), and (3) explore possible associations with the expression of immune markers Programmed cell death protein 1 (PD-1), Programmed death-ligand 1 (PD-L1), and Granzyme B (GZMB) in MCT tissue. Stool samples were collected from healthy dogs (n = 24) and dogs with MCTs (n = 24) before and after ECT and IL-12 GET. DNA was extracted from the samples, and shallow shotgun sequencing was performed. Immunohistochemistry was performed on the tumors to assess the expression of PD-1, PD-L1, and GZMB. The dysbiosis index, alpha diversity, and beta diversity did not differ between groups. Regarding microbial composition, Bifidobacterium animalis, Corynebacterium variabile, Lactobacillus johnsonii, Pediococcus pentosaceus, Streptococcus anginosus, Streptococcus equinus, Streptococcus intermedius, Clostridium thermobutyricum, Megasphaera elsdenii, and Anaerobiospirillum sp. were found in lower relative abundance in feces of dogs with MCTs, while Bacteroides togonis, Lactobacillus amylolyticus, Prevotella sp. CAG:279, and Megamonas hypermegale were more abundant compared to healthy dogs. Our study provides further insight into the composition of the gut microbiota in dogs with MCTs, where ECT and IL-12 GET did not lead to major shifts. We were unable to establish any association between the expression of immune markers and the microbiota. Full article

Phospholipases A₂ (PLA₂s) are key mediators of the cytotoxic and inflammatory activities of snake venoms. While PLA₂ isoforms from Bothrops diporus venom have been characterized and shown to possess antimetastatic and antiangiogenic properties, their impact on mitochondrial bioenergetics and immune modulation has not yet been investigated. In this study, we examined the bioenergetic and immunomodulatory effects of B. diporus PLA₂s using integrated biochemical, metabolic, and multiplex cytokine analyses. In MDA-MB-231 breast cancer cells, pooled PLA₂s induced a dose-dependent decrease in MTT-reducing activity, increased mitochondrial ROS, caused Δψm hyperpolarization, and decreased NADH autofluorescence, collectively indicating sustained mitochondrial stress. Real-time impedance measurements further revealed a marked inhibition of cell proliferation. In human PBMCs, pooled PLA₂s elicited a dynamic cytokine program, inducing early cytotoxic (Granzyme B) and chemotactic (CCL2, CCL3, CCL4) mediators, followed by late pro-inflammatory and regulatory factors such as IL-6, TNF-β, IL-10 and IL-15. Analysis of a single purified PLA₂ isoform (Fraction 6) confirmed activation of the canonical IL-6/TNF-α/IL-1β axis but uniquely induced IL-10 and CCL20, revealing isoform-specific immunomodulatory properties. Altogether, these findings provide the first integrated characterization of mitochondrial and immune perturbations induced by B. diporus PLA₂s, expanding their recognized biological scope and underscoring their potential as molecular templates for novel pharmacological strategies targeting mitochondrial vulnerabilities or modulating the tumor immune microenvironment. Full article

Background/Objectives: Poly(ADP-ribose) polymerase inhibitors (PARPis) have significantly transformed the treatment landscape for ovarian cancer; however, their clinical efficacy is often limited by poor response rates and the emergence of resistance. Recent studies have revealed that in ovarian cancer cells resistant to PARPi, the expression levels of adenosine receptors are upregulated. Accumulation of adenosine activates adenosine A2A receptor (A2AR) on immune cells, leading to immune suppression and immune escape. We hypothesize that this is a key factor limiting the efficacy of PARPi and driving the development of resistance. Therefore, the rational combination of PARPi with A2AR antagonists (A2ARas) may represent a highly promising anticancer strategy. Methods: To assess the effects of the PARPi AG14361 and the A2ARa AZD4635 on ovarian cancer growth and the immune microenvironment, we conducted in vitro and in vivo experiments and utilized single-cell RNA sequencing (scRNA-seq) to construct a high-resolution immune landscape. Results: AG14361 significantly inhibited ovarian cancer growth both in vitro and in vivo, accompanied by the accumulation of cyclic adenosine monophosphate (cAMP) and activation of the cAMP/cAMP response element-binding protein (CREB) pathway in mouse cells and tumor tissues. However, compared to monotherapy, the combination of AG14361 and AZD4635 significantly enhanced antitumor activity by inhibiting cAMP accumulation and the cAMP/CREB pathway. More importantly, the combination therapy of PARPi and A2ARa reduced the infiltration of immunosuppressive cells (such as regulatory T cells and M2 macrophages) while increasing the infiltration of cytotoxic T cells and granzyme B-positive cells, thereby creating a more favorable immune microenvironment for tumor clearance. Single-cell analysis revealed distinct functional subpopulations of macrophages and T cells, highlighting the complexity of immune heterogeneity and the potential for targeting specific immune cell subpopulations to enhance therapeutic efficacy. Conclusions: These findings suggest that the combination therapy of PARPi and A2ARa is a highly promising strategy that overcomes PARPi-induced immune escape by targeting the cAMP/CREB axis, thereby synergistically enhancing antitumor effects and holding promise as an effective treatment for solid tumors. Full article

Neoadjuvant systemic therapy (NST) is standard for locally advanced breast cancer (BC), yet predictors of pathological complete response (pCR) remain elusive. While Natural Killer (NK) cells are vital for anti-tumor response, their specific receptor dynamics during NST are poorly defined. This study provides a high-dimensional characterization of the peripheral NK cell landscape and immune signatures associated with therapeutic success. This prospective cohort study included 34 BC patients and 35 healthy donors (HD). Clinical characteristics were collected, and peripheral blood NK cell subsets were evaluated. We utilized high-parameter flow cytometry and unsupervised clustering (UMAP) to longitudinally track NK cell phenotypes (NKG2D, DNAM-1, PD-1, TIGIT) pre- and post-NST. NK cell cytotoxicity was evaluated, and serum levels of related IL-17A (interleukin), IL-2, IL-4, IL-10, IL-6, TNF-α (tumor necrosis factor-alpha), Fas, sFasL, IFN-γ (interferon-gamma), and Granzyme A were analyzed. Patients exhibited distinct NK cell profiles according to the pathological response. Only 12 BC patients achieved pCR. These patients showed improved NK cell cytotoxicity and higher concentrations of IL-2, TNF-α, sFASL, and Granzyme B after treatment compared with Non-pCR patients. In contrast, in Non-pCR patients, the percentages of CD56^bright NK cells increased after neoadjuvant therapy, whereas the more cytotoxic CD56^dim NK cell population decreased. Additionally, NK cells from Non-pCR patients exhibited higher co-expression of inhibitory checkpoints (TIGIT and PD-1), indicating reduced NK cell function. Otherwise, pCR patients displayed a more favorable balance of activating receptors (NKG2D and DNAM-1), and a favorable shift in the TIGIT/DNAM-1 activating-to-inhibitory axis. This study highlights the potential role of NK cells in determining the response to neoadjuvant therapy in BC patients. Those who achieved pCR showed enhanced NK cell activity and higher expression of activating receptors. Moreover, NK cells from Non-pCR patients showed lower cytotoxicity and higher expression of inhibitory receptors. These results suggest that NK cell phenotype evaluation could serve as a biomarker of treatment response in patients with BC. They also showed that the TIGIT/DNAM-1 axis can be a critical determinant of pCR. Full article