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BACKGROUND/OBJECTIVES: Natural killer (NK) cells are key innate lymphoid cells that restrict tumour progression by secreting proinflammatory cytokines and directly lysing malignant cells, with their activity tightly regulated by a balance of activating and inhibitory surface receptors. The natural cytotoxicity receptor NKp44 is induced on NK cells following stimulation with IL-2 or IL-15 and recognizes platelet-derived growth factor D (PDGF-DD) as a ligand. Mechanistic interpretation of NKp44 signalling upon PDGF-DD engagement is confounded by the existence of three distinct NKp44 isoforms (NKp44-1, -2, and -3), each capable of initiating divergent intracellular signalling cascades. Unlike NKp44-2 and -3, NKp44-1 encodes a cytoplasmic tyrosine residue (Y238) that conforms to a putative immunoreceptor tyrosine-based inhibition motif (ITIM) and has been reported to suppress NK cell effector functions in some contexts. However, it remains unclear whether the NKp44 isoforms are translated and expressed in NK cells, and formal evidence defining NKp44-1 signalling in response to engagement by PDGF-DD is lacking. METHODS: In this study, we used C-terminal targeting monoclonal antibodies (mAbs) and a NFAT-GFP reporter system to define the expression and signalling properties of NKp44 isoforms in response to PDGF-DD. RESULTS: We demonstrate protein expression of NKp44-1 and NKp44-2-/3 receptors in IL-2 expanded NK cells. We further show that NKp44-1 transduces activating rather than inhibitory signals when engaged by PDGF-DD ligand, albeit weaker than NKp44-3. Intriguingly, we find that Y238 is dispensable for NKp44-1 activating signalling and instead functions as a YXX¦µ internalisation motif. CONCLUSIONS: Collectively, these findings provide the first evidence that the NKp44-1 and NKp44-2/3 isoforms are expressed in NK cells and establish that PDGF-DD activates signalling through NKp44-1 independently of Y238. This work lays the foundations for future studies investigating how PDGF-DD sensing by the different NKp44 isoforms shapes immune functions in different physiological and pathological contexts.

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD), dexamethasone, IL©\10, or rapamycin. However, challenges remain in obtaining robust and efficient generation of cell therapy©\based tolDCs without compromising their viability. We recently reported that CCR2©\dependent recruitment of monocytic cells, with the capacity to dampen T©\helper responses, occurs in mice treated with a single©\stranded oligonucleotide (ssON). Here, we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL©\4 and GM©\CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD©\L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes, many of which overlapped with VitD©\moDC and resulted in moDCs with altered lipopolysaccharide (LPS)©\responsiveness. Moreover, ssON©\moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR©\¦Ã in the generation of ssON©\moDC. Collectively, our data show that monocytes differentiated with IL©\4, GM©\CSF, and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article, the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly, the tolerogenic DCs had upregulated PD©\L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T©\helper cell responses.

Triple-negative breast cancer (TNBC) is an aggressive and clinically challenging subtype defined by the absence of estrogen receptor, progesterone receptor, and HER2 amplification, resulting in poor prognosis and limited therapeutic options. Targeting alternative molecular pathways is urgently needed to overcome resistance and improve patient outcomes. CD147 has emerged as surface marker associated with tumor progression and immune evasion. In this study, CD147 and MHC class I¡ªa key inhibitory ligand for natural killer cells¡ªwere analyzed in breast cancer cell lines (MCF7, MDA-MB-453, MDA-MB-231, and HCC38) using flow cytometry. The therapeutic efficacy of a humanized anti-CD147 monoclonal antibody (HuM6-1B9) was evaluated for its capacity to potentiate antibody-dependent cellular cytotoxicity (ADCC). HuM6-1B9 demonstrated the strong binding to MDA-MB-231 (KD?=?4.982?nM) and HCC38 (KD?=?4.523?nM), which are representative TNBC cell lines. In 3D spheroid models, HuM6-1B9 significantly enhanced PBMC-mediated ADCC, leading to a marked reduction in TNBC spheroid viability. Co-culture of CFSE-labeled MDA-MB-231 and HCC38 cells with primary NK cells confirmed robust ADCC, achieving 50% and 70% cytotoxicity, respectively, despite high MHC class I expression. Live-cell imaging demonstrated caspase-3/7 activation consistent with apoptosis in NK-targeted tumor cells, while CD107a degranulation and IFN-¦Ã secretion confirmed the functional contribution of HuM6-1B9 to ADCC enhancement. Importantly, HuM6-1B9 did not promote migration or invasion in MDA-MB-231 cells, supporting its safety profile regarding metastasis. Collectively, these findings establish HuM6-1B9 as a promising immunotherapeutic candidate that overcomes immune resistance and selectively eliminates TNBC cells through ADCC without enhancing metastatic potential. By integrating mechanistic assays of NK cytotoxicity, apoptosis, and 3D tumor spheroids, this study provides clinically relevant insights underscoring the translational potential of HuM6-1B9 in TNBC immunotherapy.